Combinations of PKC inhibitors and therapaeutic agents for treating cancers

ABSTRACT

This invention provides methods for screening protein kinase C inhibitors capable of potentiating apoptosis in tumor cells. This invention also provides methods for screening antitumor therapeutic agents suitable for combination therapy with a protein kinase C inhibitors capable of potentiating apoptosis in tumor cells. This invention further provides different combination therapies comprising the specific protein kinase C inhibitors and the antitumor therapeutic agents.

BACKGROUND OF THE INVENTION

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofeach series of experiments in this application, preceding the claims.

Protein kinase C (PKC) functions in processes relevant tocarcinogenesis, tumor cell metastasis, and apoptosis. Safingol, anoptical isomer (the L-threo enantiomer) of dihydrosphingosine, is aspecific inhibitor of PKC and may represent a novel target foranti-cancer therapy. Preclinical animal studies show that safingol alonehas minimal effects on tumor cell growth, but combination of thiscompound with conventional chemotherapy agents dramatically potentiatestheir anti-tumor effects. It has been suggested that manychemotherapeutic agents exert their anti-tumor effects by inducingapoptosis.

A large body of evidence indicates a fundamental role for theinvolvement of protein kinase C (PKC), family members ofserine/threonine protein kinases, in processes relevant to neoplastictransformation, carcinogenesis, and tumor cell invasion of surroundingtissues (1-3). Consequently, PKC may represent a novel target foranti-cancer therapy. Safingol, the L-threo enantiomer ofdihydrosphingosine, is a specific inhibitor of PKC (4). Preclinicalanimal studies show that safingol is non-toxic at doses that achieveserum levels sufficient to inhibit PKC enzyme activity (5). Whilesafingol has negligible impact on tumor cell growth in vivo, thecombination of safingol with conventional chemotherapeutic agents suchas doxorubicin and cisplatin significantly potentiates the anti-tumoreffects of these drugs (6).

Based on these observations, safingol, used in combination withdoxorubicin, has become the first PKC specific inhibitor to enterclinical trials. The mechanism by which safingol potentiates theactivity of chemotherapeutic agents is unclear, although inhibition ofP-glycoprotein phosphorylation and reversal of the multidrug resistant(mdr) phenotype have been suggested (7,8). While this hypothesis canexplain the synergism achieved with combinations of safingol anddoxorubicin, it does not explain the synergism reported for combinationsof safingol with drugs that are not believed to produce resistance bythe mdr mechanism (e.g., cisplatin) (6), nor does it explainsafingol-induced effects that occur in tumor cell lines that do notexpress the P-glycoprotein (8). Therefore, pathways other thanP-glycoprotein inhibition are likely to be involved in thesafingol-mediated enhancement of chemotherapy.

It has been suggested that the anti-tumor activity of manychemotherapeutic agents (e.g., cisplatin and etoposide) is a consequenceof their induction of apoptosis (9). In this context it has beenproposed that activation of PKC acts as an antagonist to apoptosis,whereas inhibition of PKC promotes apoptosis (10-12). Thus,safingol-mediated potentiation of chemotherapy might be attributed toits PKC inhibitory effect, subsequently leading to increased apoptosisafter drug-induced damage.

The present studies sought to determine the extent to which whethersafingol by itself, or in combination with a specific chemotherapeuticdrug (e.g. mitomycin-C, MMC), would promote apoptosis in gastric cancercells. Furthermore, applicants investigated whether the p53 status ofthese cells influences the development of apoptosis after treatment withsafingol and MMC.

SUMMARY OF THE INVENTION

This invention provides a method for screening protein kinase Cinhibitors capable of potentiating apoptosis in tumor cells comprisingsteps of (a) contacting an amount of a protein kinase C inhibitors withtumor cells effective to potentiate apoptosis of tumor cells; (b)contacting the potentiated tumor cells of step (a) with an antitumortherapeutic agent; (c) determining the apoptosis of tumor cells; and (d)comparing the apoptosis determined in step (c) with apoptosis of sametumor cells which are only treated with the antitumor therapeutic agent,an increase in apoptosis indicating that the protein kinase C inhibitoris capable of potentiating apoptosis in tumor cells. This invention alsoprovide the above method, wherein step (a) is carried out in thepresence of the antitumor therapeutic agent.

This invention also provides the protein kinase C inhibitor capable ofpotentiating apoptosis in tumor cells as determined by theabove-described methods.

This invention further provides a pharmaceutical composition comprisingthe protein kinase C inhibitor capable of potentiating apoptosis intumor cells as determined by the above-described methods and apharmaceutically acceptable carrier.

This invention provides a method for screening antitumor therapeuticagents suitable for combination therapy with a protein kinase Cinhibitors capable of potentiating apoptosis in tumor cells comprisingsteps of: (a) contacting an amount of a protein kinase C inhibitor withtumor cells effective to potentiate apoptosis of tumor cells; (b)contacting the potentiated tumor cells of step (a) with an antitumortherapeutic agent; (c) determining the apoptosis of tumor cells; and (d)comparing the apoptosis determined in step (c) with apoptosis of sametumor cells which are only treated with the protein kinase C inhibitor,an increase in apoptosis indicating that the antitumor therapeutic agentis suitable for combination therapy with a protein kinase C inhibitorcapable of potentiating apoptosis in tumor cells. In an embodiment, step(a) is carried out in the presence of the protein kinase C inhibitor. Inanother embodiment, the antitumor therapeutic agent is not previouslyknown.

This invention also provides an antitumor therapeutic agent suitable forcombination therapy with a protein kinase C inhibitors capable ofpotentiating apoptosis in tumor cells determined by the above-describedmethods.

This invention also provides a pharmaceutical composition comprising aneffective amount of the antitumor therapeutic agent determined by theabove-described methods, a protein kinase C inhibitor capable ofpotentiating apoptosis in tumor cells and a pharmaceutically acceptablecarrier.

This invention provides a method for enhancing therapy in a tumorbearing subject comprising administering to the subject an effectiveamount of a specific protein kinase C inhibitor capable of potentiatingapoptosis in tumor cells during or prior to the treatment of anantitumor therapeutic agent. This invention provides the above method,wherein the specific protein kinase C inhibitor is Safingol(L-threo-dihydrosphingosine), RO32-0432 (Bisindolylmaleimide tertiaryamine), UCN-01 (7-OH-staurosporine), Flavopiridol (L86-8275), Bryostatin1 (Macrocyclic lactone) or antisense nucleotides capable of inhibitingthe expression of the protein kinase C.

In an embodiment, the antitumor therapeutic agent is a chemotherapeuticagent. In a further embodiment, the chemotherapeutic agent is selectedfrom a group consisting of Mitomycin C, Carboplatin, Taxol andDoxorubicin. In a still further embodiment, the antitumor therapeuticagent is a radiotherapeutic agent

In an embodiment of the above-described methods, the tumor is agastrointestinal cancer. In a further embodiment, the gastrointestinalcancer is gastric cancer, small bowel cancer, colon cancer or rectalcancer.

In a separate embodiment of the above-described methods, the tumor is abreast cancer. In another embodiment, the tumor is a ovarian cancer. Ina still another embodiment, the tumor is of prostate cancer, lungcancer, melanoma, cervical carcinoma, pancreatic cancer, sarcoma,hepatoma, gallbladder cancer, leukemia or lymphoma.

Finally, this invention provides a method for potentiating apoptosis intumor cells comprising contacting the cancerous cells with an effectiveamount of a specific protein kinase C inhibitor capable of potentiatingapoptosis during or prior to the treatment of an antitumor therapeuticagent.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Photomicrographs of representative fields of SK-GT-5 cells(mutant for p53) stained with bibenzimide trihydrochloride(Hoescht-33258) to evaluate nuclear chromatin condensation (i.e.,apopotosis) after treatment for 24 hours with no drug (panel A),safingol alone (50 μM, panel B), MMC alone (5 μg/ml, panel C), or thecombination of safingol (50 μM) and MMC (5 μg/ml) (panel D).

FIG. 2. Induction of apoptosis in MKN-74 cells (a wild-type for p53)with safingol alone (50 μM), MMC alone (5 μg/ml), or safingol (50 μM) incombination with MMC (5 μg/ml). Cells were counted and scored forapoptotic chromatin condensation by the QFM method described in the"Material and Methods" section. Bars represent mean number of cellscounted that undergo apoptosis as a percentage of 500 total cellsrandomly counted in duplicate samples, ±SD.

FIG. 3. Contour plots are shown for the frequency of MKN-74 cellsexhibiting increased green fluorescence emitted fromdigoxigenin-dUTP:fluorescein isothyiocyanate-labeled antibody complexesdUTP Incorp. (FITC fluorescence)!, which identifies subpopulations ofcells undergoing apoptosis by the TdT assay, versus cellular DNA content(PI fluorescence), as described in the "Material and Methods" section.MKN-74 cells were incubated for 24 hours with no drug (panel A),safingol alone (50 μM, panel B), MMC alone (5 μg/ml, panel C), or thecombination of safingol (50 μM) and MMC (5 μg/ml) (panel D). Apoptoticcells are identified as those sorting above the R₁ region of the contourplots. The G₀ /G₁, S, and G₂ /M regions of the cell cycle are indicatedin Panel A.

FIG. 4. Effect of PKC activation with PMA on the induction of apoptosisin the presence of the PKC inhibitor safingol. SK-GT-5 cells weretreated, as described in "Materials and Methods," according to thefollowing protocols: safingol alone (50 μM) for 1 hour followed by nodrug for 24 hours; MMC alone (5 μg/ml) for 24 hours; safingol alone (50μM) for 1 hour followed MMC (5 μg/ml) alone for 24 hours; PMA alone (1ng/ml) for 1 hour, followed by no drug for 24 hours; and the combinationof safingol (50 μM) and PMA (1 ng/ml) for 1 hour, followed by MMC alone(5 μg/ml) for 24 hours. Induction of apoptosis was then determined withthe QFM staining of condensed nuclear chromatin. Bars represent meannumber of cells counted that undergo apoptosis as a percentage of 500total cells randomly counted in duplicate samples, ±SD.

FIG. 5. Treatment with safingol alone did not induce significant levelsof apoptosis (2%±1) when compared with untreated controls (<1%).

FIG. 6. UCN-01 enhances MMC-induced apoptosis: SK-GT-2 cells, which havea mutation in p53, were treated with increasing concentrations of UCN-01in the presence or absence of a fixed dose of MMC for 24 hours. Theresults for the QFM analysis are shown in FIG. 6.

FIG. 7. Apoptosis was again measured by QFM staining. Applicants'results are shown in FIG. 7.

Applicants' studies indicate that flavopiridol increased the inductionof apoptosis from 7%±1% with MMC alone to 73%±1% with MMC andflavopiridol in combination. Flavopiridol alone induced a slight degreeof apoptosis (17%±2%), but not nearly to the degree observed with thecombination therapy.

FIG. 8. Each lane represents the following: lane A, the IgG control;lane B, no drug therapy for 24 hours; lane C, MMC alone (5.0 μg/ml) for24 hours; lane D, safingol alone (50 μM) for 24 hours; lane E, thecombination of safingol (50 μM) and MMC (5.0 μg/ml) for 24 hours. Theresults indicate that untreated gastric cancer cells (lane B) haveincreased cdk2 activity and that both MMC (lane C) and safingol (lane D)have decreased cdk2. However, the combination of MMC and safingolincreased cdk2 activity back to basal levels.

FIG. 9. MMC alone at the concentrations of 2.5 μg/ml and 5.0 μg/mlinduced apoptosis in 4%±2 and 6%±1 of the MDA-MB-468 cells,respectively. However, the combination of safingol (SPC) and MMCsignificantly increased the percentage of cells undergoing apoptosisfrom 11%±1 with safingol alone to 23%±2 with safingol and 2.5 μg/ml MMCand to 33%±10 with safingol and 5.0 μg/ml MMC (p<0.001).

FIG. 10. The combination of UCN-01 and MMC together increased theinduction of apoptosis of the MDA-MB-468 cells from 20%±4 with UCN-01alone to 41%±3 with UCN-01 and 2.5 μg/ml MMC and to 58%±1 with UCN-01and 5.0 μg/ml MMC.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a method for screening protein kinase Cinhibitors capable of potentiating apoptosis in tumor cells comprisingsteps of (a) contacting an amount of a protein kinase C inhibitor withtumor cells effective to potentiate apoptosis of tumor cells; (b)contacting the potentiated tumor cells of step (a) with an antitumortherapeutic agent; (c) determining the apoptosis of tumor cells; and (d)comparing the apoptosis determined in step (c) with apoptosis of sametumor cells which are only treated with the antitumor therapeutic agent,an increase in apoptosis indicating that the protein kinase C inhibitoris capable of potentiating apoptosis in tumor cells.

This invention also provide the above method, wherein step (a) iscarried out in the presence of the antitumor therapeutic agent. In anembodiment, the protein kinase C inhibitor is not previously known.

This invention also provides the protein kinase C inhibitor capable ofpotentiating apoptosis in tumor cells as determined by theabove-described methods.

This invention further provides a pharmaceutical composition comprisingthe protein kinase C inhibitor capable of potentiating apoptosis intumor cells as determined by the above-described methods and apharmaceutically acceptable carrier.

This invention provides a method for screening antitumor therapeuticagents suitable for combination therapy with a protein kinase Cinhibitors capable of potentiating apoptosis in tumor cells comprisingsteps of: (a) contacting an amount of a protein kinase C inhibitor withtumor cells effective to potentiate apoptosis of tumor cells; (b)contacting the potentiated tumor cells of step (a) with an antitumortherapeutic agent; (c) determining the apoptosis of tumor cells; and (d)comparing theaapoptosis determined in step (c) with apoptosis of sametumor cells which are only treated with the protein kinase C inhibitor,an increase in apoptosis indicating that the antitumor therapeutic agentis suitable for combination therapy with a protein kinase C inhibitorcapable of potentiating apoptosis in tumor cells. In an embodiment, step(a) is carried out in the presence of the protein kinase C inhibitor. Inanother embodiment, the antitumor therapeutic agent is not previouslyknown.

This invention also provides an antitumor therapeutic agent suitable forcombination therapy with a protein kinase C inhibitors capable ofpotentiating apoptosis in tumor cells determined by the above-describedmethods.

This invention also provides a pharmaceutical composition comprising aneffective amount of the antitumor therapeutic agent determined by theabove-described methods, a protein kinase C inhibitor capable ofpotentiating apoptosis in tumor cells and a pharmaceutically acceptablecarrier.

This invention provides a method for enhancing therapy in a tumorbearing subject comprising administering to the subject an effectiveamount of a specific protein kinase C inhibitor capable of potentiatingapoptosis in tumor cells during or prior to the treatment of anantitumor therapeutic agent. This invention provides the above method,wherein the specific protein kinase C inhibitor is selected from a groupconsisting of Safingol (L-threo-dihydrosphingosine), RO32-0432(Bisindolylmaleimide tertiary amine), UCN-01 (7-OH-staurosporine),Flavopiridol (L86-8275), Bryostatin 1 (Macrocyclic lactone) andantisense nucleotides capable of inhibiting the expression of theprotein kinase C. The above specific protein kinase C inhibitors aresimply described for illustrative purposes of this invention. As it willbe appreciated by a person of ordinary skill in the art that otherspecific protein kinase inhibitors may be used in this invention.Applicants have provided as methodology to determine whether a proteinkinase C inhibitor may be useful for the claimed combination therapy.

In an embodiment, the antitumor therapeutic agent is a chemotherapeuticagent. In a further embodiment, the chemotherapeutic agent is MitomycinC, Carboplatin, Taxol or Doxorubicin. In a still further embodiment, theantitumor therapeutic agent is a radiotherapeutic agent

In an embodiment of the above-described methods, the tumor is agastrointestinal cancer. In a further embodiment, the gastrointestinalcancer is gastric cancer, small bowel cancer, colon cancer or rectalcancer, leukemia or lymphoma.

In a separate embodiment of the above-described methods, the tumor is abreast cancer. In another embodiment, the tumor is a ovarian cancer. Ina still another embodiment, the tumor is selected from a groupconsisting of prostate cancer, lung cancer, melanoma, cervicalcarcinoma, pancreatic cancer, sarcoma, hepatoma and gallbladder cancer.

Finally, this invention provides a method for potentiating apoptosis intumor cells comprising contacting the cancerous cells with an effectiveamount of a specific protein kinase C inhibitor capable of potentiatingapoptosis during or prior to the treatment of an antitumor therapeuticagent.

This invention will be better understood from the Experimental Detailswhich follow. However, one skilled in the art will readily appreciatethat the specific methods and results discussed are merely illustrativeof the invention as described more fully in the claims which followthereafter.

EXPERIMENTAL DETAILS

First Series of the Experiments

Materials and Methods

Cell culture

Early passage human gastric cancer cell lines SK-GT-5 and MKN-74 wereestablished and characterized as described previously (13,14). Allcultures were maintained at 37° C. in Eagles minimum essential media(MEM) supplemented with 20% fetal calf serum in an humidified 5% CO₂atmosphere; the cultures tested negative for Mycoplasma species.

Determination of Apoptosis

Apoptosis was measured by two different methods: (i) Quantitativefluorescent microscopy (QFM) of nuclear changes induced by apoptosis, asdetermined by bisbenzimide trihydrochloride (Hoescht-33258) staining ofnuclear chromatin (15), and (ii) a terminal deoxynucleotidyl transferase(TdT) assay that labels the 3'-OH ends of DNA fragments produced inapoptotic cells (16). Cells were treated according to one of severalprotocols: (i) no drug (control) for 24 hours; (ii) safingol alone for24 hours at 50 μM, a concentration representing the highest non-toxicdose for SK-GT cells (as determined by cell proliferation studies with ³H!-thymidine (4)) and slightly exceeding the concentration (i.e., 30 μM)which inhibits PKC enzyme activity by 50% in vitro; (iii) Mitomycin-Calone at 5 μg/ml for 24 hours; (iv) a combination of safingol (50 μM)and Mitomycin-C (5 μg/ml) for 24 hours; (v) safingol (50 μM) alone for 1hour, followed by an immediate wash out of the drug with fresh MEM, andthen an additional 24 hours of MMC (5 μg/ml) exposure; (vi) safingol (50μM) with 1 ng/ml 3-phorbol 12-myristate 13-acetate (PMA) for 1 hour,followed by an immediate wash out of the drug with MEM and 24 hours ofMMC (5 μg/ml) exposure; and (vii) either safingol (50 μM) alone or PMA(1 ng/ml) alone for 1 hour, followed by drug washout with MEM and 24hours of exposure to media without drug. The dose of MMC used was basedon proliferating studies with ³ H!-thymidine (14, and unpublished data)indicating 20% inhibition of cell proliferation. MMC was diluted inwater. A stock safingol solution was constituted in DMSO and wassubsequently diluted at least 1:100 in media for all experiments. Thisdilute concentration of DMSO does not induce apoptosis or inhibit cellproliferation in these cell lines (unpublished data). For QFMdeterminations, the cells were fixed in 3% paraformaldehyde andincubated at room temperature for 10 minutes. The fixative was removedand the cells were washed with 1× PBS, resuspended in 20 μl 1× PBScontaining only 8 μg/ml of bisbenzimide trihydrochloride (Hoechst#33258), and incubated at room temperature for 15 minutes. Aliquots ofthe cells (10 μl) were placed on glass slides coated with3-amino-propyl-triethoxysilane, and duplicate samples of 500 cells eachwere counted and scored for the incidence of apoptotic chromatincondensation using an Olympus BH-2 fluorescence microscope equipped witha BH2-DM2U2UV Dich. Mirror Cube filter (Olympus, Lake Success, N.Y.).For the TdT assays, the ApopTag Kit (Oncor, Gaithersburg, Md.) was used.This method employs a fluoresceinated antidigoxigenin antibody directedagainst nucleotides of digoxigenin-11-dUTP (d-dUTP) which arecatalytically added to the 3-ends of fragmented DNA by TdT. Briefly,1-2×10⁶ cells were washed and fixed with 1% paraformaldehyde. The fixedcells were incubated in a reaction mixture containing TdT and d-dUTP for30 minutes at 37° C. Stop/wash buffer was added, and the cells wereresuspended in 100 μl of fluorescinated anti-digoxigenin antibody for 30minutes at room temperature. The cells were then washed with 0.1% TritonX-100 and counterstained with propidium iodide (PI) solution. Green(d-dUTP labeled DNA strand breaks) and red (PI staining for total DNAcontent) fluorescence of individual cells were measured on a FACScanflow cytometer (Becton Dickinson, San Jose, Calif.). The resultingbivariate plots enabled the detection of apoptotic events within thecell cycle. The R₁ cursor was set using the control specimen to definenormal levels of green fluorescence (i.e., basal levels of apoptosis).Cells with fluorescence above the R₁ cursor were considered apoptotic.The data from 10,000 cells were collected and analyzed using CellFit andLYSYS software (Becton Dickinson).

Statistical analysis

All experiments were done in duplicate and were repeated at least threetimes unless otherwise indicated. The statistical significance of theexperimental results was determined by the two-sided Student's t test.

EXPERIMENTAL RESULTS

Effect of safingol and MMC on inducing apoptosis in SK-GT-5 and MKN-74cells.

SK-GT-5 cells, which have a mutated p53 gene (14) and have deficient p53function (unpublished data), were treated with safingol in the presenceor absence of MMC for 24 hours. Cells were counted and scored forcondensed nuclear chromatin, and representative photomicrographs areshown in FIG. 1. Treatment with safingol alone (Panel B) did not inducesignificant levels of apoptosis (2%±1) when compared with untreatedcontrols (<1%, Panel A). However, the combination of safingol and MMC(Panel D) significantly increased the percentage of cells undergoingapoptosis from 18%±1 with MMC alone (Panel C) to 39%±1 with the drugcombination (p <0.001). Treatment of SK-GT-5 cells with safingol foronly 1 hour prior to 24 hours of treatment with MMC resulted in anincrease in apoptosis (44%±4% of exposed cells) that was essentiallyequivalent to that which was observed with concomitant exposure of thecells to the two drugs for the entire 24 hour interval (data not shown).MKN-74 cells, which are a wild- type for p53, were subjected to the sametreatments. As shown in FIG. 2, cell counts of MKN-74 cells followingtreatment with safingol alone indicated no significant induction ofapoptosis (8%±3) when compared to untreated controls (<1%). However, thecombination of safingol and MMC significantly increased the percentageof cells undergoing apoptosis from 40%±4 with MMC alone to 83%±4 withsafingol and MMC together (p<0.005).

Identifying gastric cancer cells undergoing apoptosis by the TdT assay

MKN-74 cells were treated in the same manner and analyzed by combiningthe TdT assay and flow cytometry. With this approach, apoptotic cellswere identified as those shifting above the R₁ region of the contourplot. FIG. 3 shows that MKN-74 cells incubated with safingol alone for24 hours (Panel B) had the same levels of green fluorescence as thecontrol cells (Panel A), i.e., essentially no apoptosis. Treatment withMMC alone for 24 hours (Panel C) resulted in the cells exhibitingincreased green fluorescence (sorted above the R₁ region), indicatingdevelopment of apoptosis. The results in Panel C indicate that theapoptotic cells, after MMC treatment, alone are derived from those thatwere in the G₀ /G₁ and S phases of the cell cycle. However, exposure ofthe MKN-74 cells to the combination of MMC and safingol for 24 hours(Panel D) resulted in an even greater increase in cells exhibitingelevated green fluorescence. In the presence of safingol and MMC,apoptotic cells appear to arise out of all phases of the cell cycle.Similar results were also observed for SK-GT-5 cells (data not shown).

Effect of PMA on apoptosis induced by safingol and MMC

PMA activates PKC by binding to its regulatory site (17). Safingol, as asphingosine, inhibits PKC activity by interfering with the function ofPKC's regulatory domain (18). Therefore, applicants hypothesized that ifthe potentiation of MMC-induced apoptosis by safingol is mediated byinhibition of PKC, then PMA should abrogate this effect. As shown inFIG. 4, treatment of SK-GT-5 cells with either safingol or PMA alone hadessentially no effect on inducing apoptosis. Preexposure of the cells tosafingol alone for one hour followed by MMC for an additional 24 hoursinduced a significant increase in apoptosis from 36%±4 with a 24 hourexposure of MMC alone to 55%±1 with the combination (p<0.01). However,under the conditions in which cells were preexposed to PMA and safingoltogether for one hour before MMC, the induction of apoptosis wasinhibited to the level observed for MMC alone.

Experimental Conclusion

The present studies show that safingol potentiates the cytotoxic effectof MMC in two human gastric cancer cell lines which differ in theirbaseline sensitivity to MMC and in their p53 status. Neither cell lineexpresses P-glycoprotein (unpublished data). As shown by bisbenzimidetrihydrochloride staining and the TdT assay, safingol alone did notdramatically induce apoptosis in either the MMC-sensitive MKN-74 cells,which have a wild-type p53 function, or the MMC-resistant SK-GT-5 cells,which are deficient for p53. In addition, the typical oligonucleosomalbase pair fragments (DNA "ladders") were not induced by safingol underthe conditions tested (data not shown). When exposed to MMC alone,apoptosis was induced in both cell lines although to different degrees.Addition of safingol potentiated this apoptotic response approximately2-fold in both cell lines. Applicants have observed a similarpotentiation of apoptosis by safingol with other chemotherapeuticagents, including doxorubicin.

Recent studies have suggested a link between the induction of apoptosisand a p53-dependent pathway (19). Cells which express wild-type p53 arecapable of undergoing apoptosis after exposure to commonchemotherapeutic agents or ionizing radiation; whereas cells withmutated or deleted p53 are resistant, avoiding apoptosis and continuingto replicate. In view of the prevalence of p53 mutations in solidtumors, overcoming this form of resistance should greatly enhance theefficacy of cancer chemotherapy. Applicants' studies demonstrate thatsafingol sensitizes gastric cancer cells, with either wild-type ormutated p53, to the induction of apopotosis by MMC. These resultssuggest that the effect of safingol on enhancing MMC-induced apoptosismay be independent of the p53 status of the cells. The existence of ap53- independent pathway for growth arrest has been reported (20).Further studies to define the effects of safingol, especially as itpertains to PKC and steps independent of p53 in the cell cycle, are asubject of ongoing investigation.

The mechanism of action of safingol may be associated with its anti-PKCeffect. Safingol is a highly specific inhibitor of PKC (4). Safingolinhibits PKC activity by binding to its amino-terminal regulatory domain(18), which contains PKC's phorbol ester binding site (17). PKCinhibitors, such as staurosporine, inhibit PKC activity presumably bybinding to the catalytic domain at the C-terminus of PKC (21). The useof staurosporine has been associated with a high level of nonspecifictoxicity due to the fact that the catalytic domain of PKC is highlyhomologous to similar domains found in other protein kinases (e.g.,pp60^(v-src), cAMP-dependent protein kinase A, and casein kinase), eachof which is critical for normal cellular functions (21,22). In contrast,safingol shows no toxicity in vivo at concentrations that effectivelyinhibit PKC (5). The fact that PMA, which competes with safingol for theregulatory binding site of PKC, effectively abrogated the safingoleffect of potentiating MMC-induced apoptosis, supports the hypothesisthat this process is a PKC-dependent event.

Sphingosines, including dimethylsphingosine, have been reported toinduce DNA fragmentation as single agents (23). In applicants' studies,safingol (L-threo-dihydrosphingosine) alone did not induce apoptosiswhen administered to gastric cancer cells. This observation wasconfirmed by histochemical staining, the TdT assay, and the failure toinduce DNA "ladders" under identical experimental conditions. The reasonfor the different effects of sphingoid bases on inducing apoptosisremains unexplained. The different results may depend on whichenantiomer of sphingosine is used, since it appears that differentsphingosine enantiomers induce varying physiological responses. Forexample, D-erythro-sphingosine induces dephosphorylation of theretinoblastoma gene product in human T-cells; whereas the racemicmixture of DL-threo-sphingosine does not (24). Thus, the actualsphingosine used may be critical in evaluating its ability to induceapoptosis. This hypothesis is currently being tested. Nevertheless, theinability of safingol alone to induce apoptosis supports the in vivoobservation of the lack of single agent activity against tumorxenografts. The anti-cancer effect of safingol in vivo depends onconcomitant exposure with a standard chemotherapeutic agent.

Recent investigations into the elements that regulate apoptosis haveprovided evidence for the existence of a balance between pro- andanti-apoptotic signaling that determines the final choice. This balanceappears to be reciprocally regulated through the sphingomyelin signaltransduction pathway that mediates the pro-apoptotic signals (15,25) andthe activation of the phosphoinositide-PKC pathway that mediates theanti-apoptotic signals (26,27). Thus, inhibition of thephosphoinositide-PKC pathway by the PKC-specific inhibitor safingol incombination with MMC may be sufficient to tip the balance in favor ofpro-apoptotic signals.

The demonstration that safingol potentiates chemotherapy-inducedapoptosis, presumably via inhibition of PKC, may have important clinicalimplications in view of its recent introduction into clinical trials asa specific PKC inhibitor, in combination with chemotherapy. This agentis well-tolerated, and serum levels of safingol can be achieved inpatients approximating those that potentiate the effects of chemotherapyin tumor-bearing animals (28).

While alternative mechanisms for safingol action may exist (i.e.,inhibition of DNA repair), applicants' studies indicate that safingolmay represent a new class of therapeutic agents that potentiate thecytotoxic effects of anti-cancer chemotherapy through the inhibition ofPKC and the enhancement of apoptosis. Of particular importance is thefinding that safingol is effective even in tumor cells that have amutated p53 and exhibit relative resistance to chemotherapy. Hence, theuse of safingol may provide a new approach to overcoming drug resistancein tumors that are resistant to chemotherapy by virtue of lacking p53function.

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10. Jarvis W. D., Turner A. J., Povirk L. F., Traylor R. S., and GrantS. Induction of apoptotic DNA fragmentation and cell death in HL-60human promyelocytic leukemia cells has been reported for pharmacologicalinhibitors of protein kinase C. Cancer Res. 54: 1707-1714, 1994.

11. McConkey D. J., Hartzell P., Jondal M., and Arrenius S. Inhibitionof DNA fragmentation in thymocytes and isolated thymocyte nuclei byagents that stimulate protein kinase. J. Biol. Chem. 264: 13399-13402,1989.

12. Haimovitz-Friedman A., Balaban N. A., McLoughlin M., Ehleiter D.,Michaeli J., Vlodavsky I., and Fuks Z. PKC mediates basic fibroblastgrowth factor of endothelial cells against radiation-induced apoptosis.Cancer Res. 54: 2591-2597,1994.

13. Altorki N., Schwartz G. K., Blundell M., Davis D., Kelsen D., andAlbino A. Characterization of cell lines established from humangastric-esophageal adenocarcinomas: Biologic phenotype and invasionpotential. Cancer 72: 649-657, 1993

14. Nabeya Y., Loganzo F., Maslak P., Lai L., et al. The mutationalstatus of p53 protein in gastric and esophageal adenocarcinoma celllines predicts sensitivity to chemotherapeutic agents. Int. J. Cancer64: 1-10, 1994.

15. Haimovitz-Friedman A., Chu-Cheng K., Ehleiter D., Persaud R. S. etal. Ionizing radiation acts on cellular membranes to generate ceramideand initiate apoptosis. J. Exp. Med. 180: 525-535, 1994.

16. Schmitz G., Walter T., Serbel R., and Kessle C. Nonradioactivelabeling of oligonucleotides in vitro with the hapten digoxigenin bytailing with terminal transferase. Anal. Biochem. 192: 222-231, 1991.

17. Blumberg P. M. Protein kinase C as the receptor for the phorbolester tumor promoter: Sixth Rhoads memorial award lecture. Cancer Res.48: 1-8, 1988.

18. Hannun Y. A., Loomis C. R., Merrill A. H., and Bell R. M.Sphingosine inhibition of protein kinase C activity and of phorboldibutyrate binding in vitro and in human platelets. J. Biol. Chem. 261:12602-12609, 1986.

19. Lowe S. W., Ruley H. E., Jack T., Housman D. E. p53-Dependentapoptosis modulates the cytotoxicity of anticancer agents. Cell 74:957-967, 1993.

20. Johnson M., Dimitrov D., Vojta P. J., Barrett J. C., Noda A.,Pereira-Smith O., and Smith J. Evidence for a p53-independent pathwayfor upregulation of SDI1/CIP1/WAF1/p21 RNA in human cells. MolCarcinogenesis 11: 59-64, 1994.

21. Tamaoki T. and Nakano H. Potent and specific inhibitors of PKC ormicrobial origin. Biotech. 8: 732-735, 1990.

22. Nakano H., Kobayashi, E., Takahashi, I., Tamaoki, T., Kuzuu, Y., andIba, H. Staurosporine inhibits tyrosine-specific protein kinase activityof Rous sarcoma virus transforming protein. J. Antibiot (Tokyo)40:706-708, 1987.

23. Ohta H., Sweeney E. A., Masamune A., Yatomi Y., Hakomori S., andIgarashi Y. Induction of apoptosis by sphingosine in human leukemicHL-60 cells: a possible endogenous modulator of apoptotic DNAfragmentation occurring during phorbol ester-induced differentiation.Cancer Res. 55: 691-697, 1995.

24. Choa R., Khan W., and Hannun Y. A. Retinoblastoma proteindephosphorylation induced by D-erythro-sphingosine. J. Bio. Chem. 267:23459-23462, 1992.

25. Jarvis W. D., Kolesnick R. N., Fornari F., Traylor R. S., Gewirtz D.A. and Grant S. Induction of apoptotic DNA damage and cell death byactivation of the sphingomyelin pathway. Proc. Natl. Acad. Sci. U.S.A.91:73-77, 1994.

26. Obeid L. M., Linardic C. M., Karolak L. A., and Hannun Y. A.Programmed cell death induced by ceramide. Science 259:1769-1771, 1993.

27. Jarvis W. D., Fornari F. A., Browning J. L., Gewirtz D. A.,Kolesnick R. N., and Grant S. Attenuation of ceramide-induced apoptosisby diglyceride and pharmacological activators of protein kinase C inhuman myeloid leukemia cells. J. Biol. Chem. 268:31685-31692, 1994.

28. Schwartz G. K., Ward D., Saltz L. Casper E., et al. A phase I studyof the protein kinase C specific inhibitor safingol alone and incombination with doxorubicin. Proc. Amer. Soc. Clin. Onc. 14:1557,1995.

Second Series of Experiments

Applicants' long term objective is to improve clinical cancer therapy bydeveloping a new therapeutic strategy in the treatment of humanmalignancies which utilizes protein kinase C (PKC) as a target forenhancing chemotherapy induced apoptosis in tumor cells. Study designswill be based on applicants' pre-clinical data indicating that PKCrepresents a major new target in the enhancement of chemotherapy inducedapoptosis of human gastric cancer cells that are resistant tochemotherapy by virtue of a mutation in p53. Applicants' hypothesis isthat the activation of PKC inhibits the induction of chemotherapyinduced apoptosis and that this can be overcome by utilizing PKCinhibitors in conjunction with chemotherapy. This process may beassociated with other mechanisms including alterations in cell cycleprogression and modulation of cyclin dependent kinase 2 (cdk2) activity.Applicants' specific immediate and long term aims are:

1. To develop an integrated clinical program with PKC inhibitors incombination with chemotherapy that uses laboratory correlates (PKCactivity, cdk2 activity, and terminal deoxynucleotidyl transferase (TdT)activity in tumor tissues and leukocytes) as putative surrogateend-points of activity. The immediate goal will be to complete twocurrent phase I clinical trials with the PKC specific inhibitorL-threo-dihydrosphingosine (safingol) in combination with (i)doxorubicin (DOX) and (ii) cisplatin with the plan to take this to aphase II study in gastric cancer; and to initiate additional clinicaltrials with combinations of chemotherapy and other PKC inhibitorsincluding UCN-01, flavopiridol, and bryostatin.

2. To perform laboratory studies with human gastric, breast, and ovarycancer cells to: i) study a variety of new PKC inhibitors currentlyunder pre-clinical development as inducers of apoptosis in combinationwith chemotherapy; ii) define the optimal conditions in which to combinesafingol, UCN-01, flavopiridol, and bryostatin with chemotherapy forfuture clinical development; and iii) further examine cdk2 in vitro andto identify other related cell cycle dependent proteins associated withinduction of apoptosis and the inhibition of PKC.

3. To determine whether a specific PKC isoform may be a critical targetfor drug development in induction of apoptosis with chemotherapy by: (i)testing whether an antisense for PKCα in combination with MMC hassuperior anti-tumor activity against human gastric cancer cells whencompared to either agent alone or to a PKC missense with MMC; (ii)testing PKC antisense against other PKC specific isoforms in combinationwith chemotherapy; (iii) studying in gastric cancer cells whether any ofthe PKC isoforms are being preferentially inhibited during the inductionof apoptosis.

Background

Recent studies have indicated a link between the induction of apoptosis(programmed cell death) and p53 expression (1). Cells which expresswild-type p53 are capable of undergoing apoptosis after exposure tocommon chemotherapeutic agents or ionizing radiation; whereas cells withmutated or deleted p53 are resistant, avoiding apoptosis and continuingto replicate. A major hurdle for cancer chemotherapy is how to overcomethis form of drug resistance. It has been suggested that the anti-tumoractivity of many chemotherapeutic agents (e.g., cisplatin and etoposide)is a consequence of their induction of apoptosis (1,2). Recentinvestigations into the elements that regulate apoptosis have providedevidence for the existence of a balance between pro- and anti-apoptoticsignaling that determines the final choice. Applicants' hypothesis isthat this balance appears to be reciprocally regulated through thesphingomyelin signal transduction pathway that mediates thepro-apoptotic signals (3,4) and the activation of thephosphoinositide-Protein Kinase C (PKC) pathway that mediates theanti-apoptotic signals (5-7). Thus, inhibition of thephosphoinositide-PKC pathway by the PKC specific inhibitors may besufficient to tip the balance in favor of pro-apoptotic signals.Consequently, PKC may represent a novel target for anti-cancer therapyin gastric cancer.

In the United States adenocarcinoma of the stomach is the 8th leadingcause of death from cancers. It is estimated that over 24,000 new caseswill be diagnosed in 1995 and this will be associated with approximately14,000 deaths (8). Even though the number of newly diagnosed patientswith gastric cancer in the United States has remained essentiallyconstant over the past 10-15 years, the incidence of proximal gastriccancers, which is a much more aggressive form of this disease, appearsto be rising rapidly in this country (9). This disease appears to affectyoung males and its incidence is independent of socio-economic status.There have been no identifiable factors to account for the dramatic risein tumors of the gastric cardia or gastro-esophageal junction, but therate of rise exceeds that of lung cancer or melanoma for this age group.Adjuvant chemotherapy for this disease has not had a meaningful impacton survival (10) and in the metastatic setting objective response ratesare low and generally brief.

The tumors of many of the patients with gastric cancer are resistant tothe most active chemotherapeutic agents. The basis of drug resistance inthese disease has not been well defined. The expression of themulti-drug resistant gene (mdr1), which encodes the P-glycoprotein, islow (11), suggesting alternate mechanisms of drug resistance. Forgastric cancer applicants have been successful in establishing fivelong-term cell lines, SK-GT-1, SK-GT-2, SK-GT-4, SK-GT-5, and SK-GT-6for Sloan Kettering Gastric Tumor (12,13) none of which expressP-glycoprotein (14). Yet, all these tumors are resistant to chemotherapyand to the induction of apoptosis. Applicants have reported that therelative sensitivity of these gastric cancer cells to chemotherapy isinfluenced by their p53 status (14). These results are summarized belowwith the relative resistance (% cell survival by clonogenic assay/% cellsurvival of MKN-74 cells, wild-type for p53, by clonogenic assay) to 10μg/ml Cisplatin (CDDP), 5 μg/ml Mitomycin-C (MMC), and 50 μM5-Fluorouracil (5-FU) noted:

    ______________________________________                                        Fold-Increase in Resistance                                                   Relative to MKN-74 (p53.sup.wt)                                                          p53                                                                           mutational                                                         Cell line/ status/                                                            site       codon       CDDP     MMC    5-FU                                   ______________________________________                                        SK-GT-1/   17 base pair                                                                              21X      166X   11X                                    GE         deletion/57-                                                       junction   62                                                                 SK-GT-2/   CGC to CAC/ 11X      106X   12X                                    fundus     175                                                                SK-GT-4/   CGC to CAC/ N.A.*    N.A.   N.A.                                   GE         175                                                                junction                                                                      SK-GT-5/   GAC to GAG/ 12X      55X    6X                                     GE         281                                                                junction                                                                      MKN-74/    No mutation 1X       1X     1X                                     lymph node                                                                    NU-GC-4/   No mutation 2X       10X    0.6X                                   lymph node                                                                    ______________________________________                                         *Not assayed                                                             

Applicants' results indicate that the that the MKN-74 and the NU-GC-4cells, which are wild-type for p53, are exhibit sensitivity tochemotherapy (e.g. CDDP, 5-FU, and MMC) and to the induction ofapoptosis; whereas the SK-GT cell lines which have a mutation in p53exhibit resistance to chemotherapy and to the induction of apoptosis(14).

Since the activity of PKC has been reported to be increased in gastriccancer cells (15) and since p53 mutation is prevalent in gastric tumors(16), applicants believed applicants could use these gastric cancer celllines to test applicants' hypothesis that the activation of PKC inhibitsthe induction of chemotherapy induced apoptosis and that inhibiton ofPKC can enhance chemotherapy induced apoptosis even in tumor cells witha p53 mutation. The selection of a PKC inhibitor for these studiesultimately depended on its specificity for PKC. The "classic" PKCinhibitor, staurosporine (STSN), inhibits PKC activity in nanomalarconcentrations presumably by binding to the enzyme's catalytic site(17). Members of the PKC family are characterized by a uniqueamino-terminal regulatory domain containing co-factor binding sites anda carboxyl-terminal catalytic domain which is homologous to that ofthese other protein kinases (18,19). However, STSN is also exceptionallytoxic (18). The concentrations of STSN that inhibit enzyme activities ofPKC, pp60^(v-src) tyrosine kinase and cAMP-dependent protein kinase A by50% (IC₅₀), as well as other enzymes that are critical for normalcellular functions, are all within a ten-fold range of concentration(17,20). The toxicity of STSN then appears to be related to itsnon-selectivity in view of the considerable homology PKC shares withthese other protein kinases (20). Due to this lack of specificity, thedevelopment of a PKC inhibitor for clinical trials will ultimatelydepend on an agent that is either highly specific for the catalyticdomain of PKC or inhibits PKC by mechanisms that are independent of thecatalytic site. A series of PKC inhibitors are now in pre-clinicaldevelopment. These agents and the site by which they inhibit PKC aresummarized in the table below:

    ______________________________________                                                      Chemical       Site of PKC                                      Drug          derivative     inhibition                                       ______________________________________                                        Safingol (5)  L-threo-       Regulatory                                                     dihydrosphingosine                                              UCN-01 (21)   7-OH-staurosporine                                                                           Catalytic                                        RO32-0432 (22)                                                                              Bisindolylmaleimid                                                                           Catalytic                                                      e tertiary amine                                                Bryostatin 1 (23)                                                                           Macrocyclic    Regulatory                                       (prolonged    lactone                                                         exposure only)                                                                Flavopiridol (24)                                                                           Flavone        Catalytic (?)                                    (in μM     L86-8275                                                        concentrations)                                                               ______________________________________                                    

Safingol, as a sphingosine (25), is a highly specific PKC inhibitory byvirtue of its interfering with the function of the enzyme's regulatorydomain (5). The concentration of safingol that inhibits PKC enzymeactivity by 50% (I₅₀) is 33 μM but exceeds 218 μM for the inhibition ofcyclic-adenosine monophosphate-dependent protein kinase A, creatininephosphokinase and casein kinase. As a single agent safingol has beenshown to have a negligible impact on tumor growth in vivo. However, thecombination of safingol with doxorubicin or cisplatin substantiallypotentiates the anti-tumor effects of these drugs (26). Based on theseobservations, safingol, used in combination with doxorubicin, has becomethe first PKC specific inhibitor to enter clinical trials, MSKCC IRBprotocols #93-44 (27), and a second trial combining safingol withcisplatin has been approved protocol, MSKCC protocol #94-115. Non-toxicdoses of safingol have been given to patients that approachconcentrations necessary to inhibit PKC in vivo and to inducechemosensitization in animals in (see Preliminary Results below).

It has been suggested that the mechanism by which safingol potentiateschemotherapeutic agents is by inhibition of P-glycoproteinphosphorylation and reversal of the multidrug resistant (mdr) phenotype(28). While this hypothesis can explain the synergism achieved withcombinations of safingol and doxorubicin, it does not explain thesynergism reported for combinations of safingol with drugs that are notbelieved to produce resistance by the mdr mechanism (e.g., cisplatin)(28), nor does it explain safingol-induced effects that occur in tumorcell lines that do not express the P-glycoprotein (29). Therefore,pathways other than P-glycoprotein inhibition are likely to be involvedin the safingol-mediated enhancement of chemotherapy. Applicantsproposed that safingol-mediated potentiation of chemotherapy might beattributed to its PKC inhibitory effect, subsequently leading toincreased apoptosis after drug-induced damage. In order to test thishypothesis applicants initially sought to determine the extent to whichsafingol by itself, or in combination with MMC would promote apoptosisin gastric cancer cells. Furthermore, applicants investigated whetherthe p53 status of these cells influences the development of apoptosisafter treatment with safingol and MMC (5).

For these initial studies applicants used the gastric cancer cell linesSK-GT-5 cells and MKN-74. Neither cell line expresses P-glycoprotein.These studies (see PRELIMINARY RESULTS) show that safingol potentiatesthe cytotoxic effect of MMC in two human gastric cancer cell lines whichdiffer in their baseline sensitivity to MMC and in their p53 status.Neither cell line expresses P-glycoprotein (unpublished data). Safingolalone did not dramatically induce apoptosis in either the MMC-sensitiveMKN-74 cells, which have a wild-type p53 function, or the MMC-resistantSK-GT-5 cells, which are deficient for p53. In addition, the typicaloligonucleosomal base pair fragments (DNA "ladders") were not induced bysafingol under the conditions tested (data not shown). When exposed toMMC alone, apoptosis was induced in both cell lines although todifferent degrees. Addition of safingol potentiated this apoptoticresponse approximately 2-fold in both cell lines. Applicants haveobserved a similar potentiation of apoptosis by safingol with otherchemotherapeutic agents, including doxorubicin.

In order to test whether the effect of safingol in inducing apoptosiswas due to its anti-PKC effect, applicants performed comparable studieswith safingol in the presence of the phorbol ester, 3-phorbol12-myristate 13-acetate (PMA), which activates PKC by binding to itsamino-terminal regulatory domain (19). Safingol inhibits PKC activity byinterfering with the function of PKC's regulatory domain (5). Therefore,applicants hypothesized that if the potentiation of MMC-inducedapoptosis by safingol is mediated by inhibition of PKC, then PMA shouldabrogate this effect. In applicants' studies with the gastric cancercells, PMA effectively abrogated the safingol effect of potentiatingMMC-induced apoptosis in this cells, supporting the hypothesis that thisprocess is a PKC-dependent event (5).

Applicants' studies demonstrate that safingol sensitizes gastric cancercells, with either wild-type or mutated p53, to the induction ofapoptosis by MMC. This suggests that the effect of safingol on enhancingMMC-induced apoptosis is independent of the p53 status of the cells. Theexistence of a p53 independent pathway for apoptosis has been reported(30). In order to identify apoptotic cells in the cell cycle applicantshave treated MKN-74 and SK-GT-5 cells with MMC and safingol and analyzedthe cells by combining the TdT assay and flow cytometry. By thisapproach applicants have reported that in the presence of safingol andMMC apoptotic cells arise out of all phases of the cell cycle (G₀ /G₁,S, and G₂ /M) (5). This is in contrast to treatment with safingol alone,in which there are no apoptotic cells detected by this method, and totreatment with MMC alone in which apoptotic cells are derived from G₀/G₁ and S (but to a significantly less degree than the combinationtherapy). Thus, it appears that safingol may effect more than one pointregulating progression through the cell cycle.

The PKC inhibitor UCN-01 has been reported to enhance anti-tumoractivity of MMC against xenografted human colon carcinoma Co-3 cells innude mice (31). Similar to safingol, UCN-01 induces apoptosis and haseffects on the cell cycle (32). Both agents induce apoptosis in allphases of the cell cycle. However, in contrast to safingol, this effectis observed with UCN-01 alone and does not require the presence ofchemotherapy (though the effect on apoptosis of combining UCN-01 withchemotherapy has not been tested). This difference between UCN-01 andsafingol may reflect the differences in cell lines tested (Jurkat Tlymphoblastic leukemia cells for UCN-01 and SK-GT gastric cells forsafingol) or it may be a direct result of the specificity of each PKCinhibitor. For example, UCN-01 is a more potent inhibitor of PKC thansafingol; whereas safingol, by virtue of interfering with PKC'sregulatory domain, is more specific. Alternatively, these PKC inhibitorsmay have different effects, either alone or in combination withchemotherapy, on cell-cycle specific events.

Progression through the cell cycle is regulated by cyclin-dependentkinases (cdks), the activation of which involves both the binding of acyclin partner and phosphorylation and dephosphorylation of specificthreonine/tyrosine residues. In particular cdk2 is activated by thephosphorylation of threonine-160 and dephosphorylation of tyrosine-15and threonine-14 (33). The activation of cdk2 complexes is necessary forthe G₁ /S progression of the cell cycle, and the inappropriateactivation of cdks has been associated with apoptosis (34). The effectof UCN-01, as a PKC inhibitor, on cdk2 activity has been studied inJurkat T leukemia cells (32). These results indicate that UCN-01increase the activity of cdk2 in association with a decrease intyrosine-15 phosphorylation. Similar studies has been performed with thePKC activator, bryostatin 1, which has undergone phase I evaluation as asingle agent (35). Short term exposure of tumor cells to bryostatininduces PKC activation with translocation of PKC to the membrane,whereas as prolonged exposure of tumor cells to bryostatin induces PKC'sinhibition by causing its depletion from the cell. It has been reportedthat treatment of U937 human monoblastoid leukemic cells with bryostatinfor less than 48 hours resulted in the inhibition of cdk2 activity withthe dephosphorylation of threonine-160; whereas treatment of these cellswith bryostatin for 72 hours or longer induced an increase in cdk2activity with increased phosphorylation (36). Thus, these two studiessupport a hypothesis that, in terms of cell cycle mediated events,apoptosis induced by PKC inhibition is associated with an increase incdk2 activity. It remains unknown whether this is secondary to decreasedexpression of cyclin inhibitors (i.e p21) that ordinarily bind to andinhibit cdk2 or whether it is a direct result of modification of thesites of cdk2 phosphorylation. The end results of these events would beto increase cdk2 activity.

PRELIMINARY STUDIES

In vitro studies indicate that safingol, UCN-01, flavopiridol, and RO32-0432 enhance MMC-induced apoptosis in gastric cancer cells:

(i) Safingol enhances MMC-induced apoptosis: SK-GT-5 cells, which have amutated p53 gene (9), were treated with safingol in the presence orabsence of MMC for 24 hours. For determination of apoptosis SK-GT-5cells were treated according to one of several conditions: (i) no drug(control) for 24 hours, (ii) Safingol alone (50 μM) for 24 hours. (Thisconcentration represents the highest non-toxic dose of safingol for theSK-GT-5 cells, as determined by cell proliferation studies, and slightlyexceeds the safingol concentration (30 μM) which inhibits PKC enzymeactivity by 50% in vitro.), (iii) Mitomycin-C alone (2.5 μg/ml) for 24hours, (iv) Mitomycin-C alone (5 μg/ml) for 24 hours, (v) thecombination of safingol (50 μM) and Mitomycin-C (2.5 μg/ml) for 24hours, (vi) the combination of safingol (50 μM) and Mitomycin-C (5μg/ml) for 24 hours. Apoptosis was measured by quantitative fluorescentmicroscopy (QFM) of nuclear changes induced by apoptosis, as determinedby bisbenzimide trihydrochloride (Hoescht-33258) staining of condensednuclear chromatin (3,5). For the QFM duplicate samples of 500 cells eachwere counted and scored for the incidence of apoptotic chromatincondensation using an Olympus BH-2 fluorescence microscope. As shown inFIG. 5, treatment with safingol alone did not induce significant levelsof apoptosis (2%±1) when compared with untreated controls (<1%):

However, the combination of safingol and MMC significantly increased thepercentage of cells undergoing apoptosis from 18%±1 with MMC alone to39%±1% with the drug combination (p<0.001). MKN-74 cells, which arewild-type for p53, were subjected to the same treatments. Cell counts ofMKN-74 cells following treatment with safingol alone indicated nosignificant induction of apoptosis (8%±3%) when compared to untreatedcontrols (<1%). However, the combination of safingol and MMCsignificantly increased the percentage of cells undergoing apoptosisfrom 40%±4% with MMC alone to 83%±4% with safingol and MMC together(p<0.005).

(ii) UCN-01 enhances MMC-induced apoptosis: SK-GT-2 cells, which have amutation in p53, were treated with increasing concentrations of UCN-01in the presence or absence of a fixed dose of MMC for 24 hours. Theresults for the QFM analysis are shown in FIG. 6.

MMC alone at the concentration 5.0 μg/ml induced apoptosis in 7%±3% ofthe SK-GT-2 cells. However, the combination of UCN-01 and MMCsignificantly increased the percentage of cells undergoing apoptosis ina dose-dependent. With 0.1 μM UCN-01 the percentage of gastric cancercells undergoing apoptosis increased from 1%±0% with UCN-01 alone to11%±2% with MMC and UCN-01 in combination. With 1 μM UCN-01, theinduction of apoptosis increased from 1%±0 with UCN-01 alone to 18%±4%with the combination (p<0.01), and with 10 μM UCN-01 the induction ofapoptosis increased from 21%±1% with UCN-01 alone to 53%±1% with thecombination therapy (p<0.001). Applicants have observed comparableeffects with the combination of UCN-01 and MMC against the breast cancercell line MDA-MB-468 which has a mutation in the p53 gene (data notshown).

(iii) Flavopiridol inhibits PKC in nanomolar concentrations and enhancesMMC-induced apoptosis: Flavopiridol is currently in phase I clinicaltrial at the NCI. In micromolar concentrations flavopiridol has beenreported to inhibit PKC activity, but in nanomolar concentrations it hasbeen reported to inhibit tyrosine kinases (24). Before testingflavopiridol's effect on enhancing MMC-induced apoptosis in applicants'cell system, applicants first elected to determine the extent to whichflavopiridol inhibited PKC activity with PKC obtained from MKN-74gastric cancer cells. For these experiments MKN-74 cells werehomogenized and PKC was purified on a DEAE cellulose column as described(37). The PKC activity was determined by determining the extent to whichthe purified PKC phosphorylated myelin basic protein in the presence orabsence of flavopiridol at 34 and 380 nM. A control sample was also runwith the PKC pseudosubstrate, which functions as an established PKCinhibitor. The results, as shown in the table below, are expressed asthe absolute counts of ³² P!ATP incorporated into myelin basicprotein/minute of assay time:

    ______________________________________                                                                   Percent                                            Condition        PKC activity                                                                            inhibition                                         ______________________________________                                        No drug          25,000    --                                                 Flavopiridol (34 nM)                                                                           4,000     84%                                                Flavopiridol (340 nM)                                                                          3,300     87%                                                Pseudosubstrate  6,000     76%                                                (positive control)                                                            ______________________________________                                    

These results indicate that, in terms of the PKC obtained from MKN-74cells, flavoperidol is an excellent PKC inhibitor. For this reasonapplicants next elected to test the effect of flavopiridol on enhancingMMC-induced apoptosis. For these studies the conditions were essentiallythe same as described above for safingol and MMC except thatflavopiridol was used at a concentration of 300 nM and MMC remainedfixed at a concentration of 50 μM. Apoptosis was again measured by QFMstaining. The results are shown in FIG. 7.

Applicants' studies indicate that flavopiridol increased the inductionof apoptosis from 7%±1% with MMC alone to 73%±1% with MMC andflavopiridol in combination. Flavopiridol alone induced a slight degreeof apoptosis (17%±2%), but not nearly to the degree observed with thecombination therapy.

(iv) RO32-0432 enhances MMC-induced apoptosis: Roche Pharmaceuticals hassupplied us with several new PKC inhibitors to test for the enhancementof chemotherapy induced apoptosis. RO 32-0432 is one of series ofbisindolylmaleimide inhibitors of PKC which are more selective thanstaurosporine (22). By introduction of a cationic sidechain and withconformational restriction of the amine sidechain, they have developedagents that are both highly PKC specific as well as orally bioavailable.For determination of apoptosis MKN-74 cells were treated according tothe same conditions described for safingol and MMC except that RO32-0432(1 μM) was substituted for safingol. Apoptosis was again measured by QFM(24). The combination of MMC with RO 32-0432 increased the percentage ofcells undergoing apoptosis from 16%±2% with MMC alone to 25%±3% with 1nM RO 32-0432 and to 41%±1% with 1 μM RO 32-0432. Similar to whatapplicants observed with the other PKC inhibitors, RO 32-0432 as asingle agent had essentially no effect on inducing apoptosis of thesecells.

(v) The combination of safingol and UCN-01 enhances MMC-inducedapoptosis to a greater degree than either agent alone with MMC: Sinceboth UCN-01 and safingol inhibit PKC by binding to two different sitesof the enzyme, applicants tested the effect that these agents hadtogether on enhancing MMC induced apoptosis of gastric cancer cells. Fordetermination of apoptosis MKN-74 cells were treated according to one ofseveral conditions: (i) no drug (control) for 24 hours, (ii) Safingolalone (50 μM) for 24 hours, (iii) UCN-01 alone (10 μM) for 24 hours,(iv) Mitomycin-C (MMC) alone (5.0 μg/ml) for 24 hours, (v) thecombination of safingol (50 μM) and UCN-01 (10 μM) for 24 hours, (vi)the combination of safingol (50 μM) and Mitomycin-C (5 μg/ml) for 24hours, (vii) the combination of UCN-01 (10 μM) and Mitomycin-C (5 μg/ml)for 24 hours, (viii) the combination of safingol (50 μM), UCN-01 (10 μM)and Mitomycin-C (5 μg/ml) for 24 hours. Apoptosis was again measured byQFM staining. The induction of apoptosis in the MKN-74 cells increasedfrom 26%±5% with MMC alone, to 36%±1% with safingol and MMC, to 60%±2%with safingol, UCN-01, and MMC in combination. The induction ofapoptosis by UCN-01 and MMC together for these cells was essentially nodifferent than MMC alone. Thus the combination of UCN-01, safingol, andMMC induced a greater degree of apoptosis than any other conditiontested in these sets of experiments.

Taken together (i-v) these results indicate that even though the PKCinhibitors safingol, UCN-01, and RO32-0432 can by themselves induce aslight degree of apoptosis in the breast and gastric cancer cells theinduction of apoptosis is greatly enhanced when the PKC inhibitors aregiven in combination with MMC. The demonstration that these agentspotentiate chemotherapy induced apoptosis may have important clinicalimplications in view of the recent introduction of at least two of theseagents into clinical trials. Furthermore indications that the effect ofcombination of the two inhibitors on enhancing MMC induced apoptosis isgreater than either PKC inhibitor alone with MMC would suggest anothernew direction for clinical development. Of particular importance is thefinding that these agents are effective even with tumor cells that havea mutation in p53. Hence, the use of PKC inhibitors may provide a newapproach to overcoming drug resistance in tumor cells that are resistantto chemotherapy because they lack p53 function. The purpose of thesestudies is then to determine how to optimize this effect in breast andgastric cancer cells by examining the best combinations of PKCinhibitors with chemotherapy as well as to examine the cellular basis ofthis phenomenon.

Safingol increases cells in S and in combination with chemotherapyincreases cdk2 activity.

(i) Safingol effects the cell cycle by increasing the percentdistribution of cells in S phase: applicants' studies indicate thatsafingol enhances chemotherapy induced apoptosis of gastric cancer cellsin all phases of the cell cycle. However, since other PKC inhibitors,including UCN-01, have been shown to have cell cycle specific activity,despite the induction of apoptosis throughout the cell cycle, applicantselected to determine whether safingol also effected cell cycle specificevents. In order to do this applicants first performed flow cytometrystudies on MKN-74 cells synchronized in G₂ /M with nocodazole and thentreated with safingol. Applicants believed that if these studies showeda perturbation in one particular phase of the cell cycle, thenapplicants could focus on a cell cycle event (e.g. cdk2) associated witha particular cell cycle phase (e.g. G₁ /S). As shown in the table below,the cells were treated according to a series of different steps (notedas a "+" in the table) resulting in a series of different conditions(listed as A-E). The cells were analyzed for their percent distributionwithin the cell cycle as determined by flow cytometry:

    __________________________________________________________________________                         SAF                                                           Nocodazole                                                                          Nocodazole                                                                          Medium                                                                            Alone,                                                                            SAF +                                                     (18 hours)                                                                          Washout                                                                             Alone                                                                             (24 hrs)                                                                          Nocod.                                                                            G.sub.1                                                                          S  G.sub.2 /M                                 Condition                                                                          0.2 μg/ml                                                                        (4 hours)                                                                           (24 hrs)                                                                          50 μM                                                                          (24 hrs)                                                                          (%)                                                                              (%)                                                                              (%)                                        __________________________________________________________________________    A    -     -     -   -   -   72.1                                                                             18.0                                                                              9.8                                       B    +     -     -   -   -    7.7                                                                             12.1                                                                             80.2                                       C    +     +     +   -   -   33.2                                                                             22.0                                                                             44.8                                       D    +     +     -   +   -   37.2                                                                             42.9                                                                             19.9                                       E    +     +     -   -   +   27.4                                                                              7.1                                                                             65.5                                       __________________________________________________________________________

As shown above, exposure of the cells to nocodazole (0.2 μg/ml) for 18hours increased the cells remaining in G₂ /M from 9.8% in untreatedcontrols (A) to 80% with the nocodazole therapy (B), consistent with acell cycle block. When cells were washed free of nocodazole for 4 hoursand then examined at that time point by flow cytometry, the percentageof cells in G₂ /M had decreased from 80% to 44.8% (indicating removal ofthe block), the percentage of cells in G₁ had increased from 7.7% to33.2% and the percentage of cells in S phase had increased from 12.1% to22.0% (C). However, if after washing out the nocodazole for four hoursthe cells were treated with 50 μM safingol (SAF) for an additional 24hours, percentage of cells in S increased to 42.9% (D). This is incontrast to the cells treated with no drug during this additional 24period. Under this condition the percentage of cells in S remained at22.0%, suggesting that safingol induced an increase in the S phasefraction. In order to determine whether safingol was inducing an arrestof cells in G₁ /S the cells were treated according to these sameconditions except that for the additional 24 hour period following theinitial nocodazole washout, the cells were exposed to both safingol andnocodazole. Applicants anticipated that if the percent increase in Sphase induced by safingol was due to cell cycle arrest then co-exposurewith nocodazole would induce the cells to remain in S and not proceedinto a G₂ /M block. As the results show (E), reexposure of the cells tonocodazole in the presence of safingol for the additional 24 hourinterval resulted in an increase in the percentage of cells in G₂ /Mfrom 19.9% with safingol alone to 65.5% with safingol and nocodazole anda decrease in the percentage of cells in S phase from 42.9% withsafingol alone to 7.1% with safingol and nocodazole.

These results suggest that safingol does not induce a cell cycle arrestat G₁ or S but may be affecting a regulatory event within the cell cyclethat promotes the induction of apoptosis once they pass through G₁ andenter S. Applicants proposed that a potential candidate for thisregulatory event was cdk2.

(ii) Safingol increases cdk2 activity only when co-administered withchemotherapy: For determination of cdk2 activity MKN-74 cells weretreated according to the conditions shown to enhance chemotherapyinduced apoptosis: (i) no drug (control) for 24 hours, (ii) Mitomycin-Calone (5.0 μg/ml) for 24 hours, (iii) Safingol alone (50 μM) for 24hours, (iv) the combination of safingol (50 μM) and Mitomycin-C (MMC,5.0 μg/ml) for 24 hours. Cdk2 activity was determined by histone type-Iphosphorylation, as described (38). For these studies cdk2 was firstimmunoprecipitated from the cell lysates following treatment under thesefour conditions. The kinase reaction was allowed to proceed at 30° for30 minutes in the presence of histone type-I and ³² P!ATP. For eachsample a control sample was run under the same conditions, but IgG wasused in place of histone Type-I. This allowed for detection onnon-specific kinase activity which was then subtracted out from the cdk2activity to determine the absolute radioactive incorporationattributable to cdk2. The samples were transferred to a 10% SDS PAGE gelfor analysis. The autoradiographs were analyzed on a phosphoimager fordeterminations of relative cdk2 activity. The results are shown in FIG.8

Each lane represents the following: lane A, the IgG control; lane B, nodrug therapy for 24 hours; lane C, MMC alone (5.0 μg/ml) for 24 hours;lane D, safingol alone (50 μM) for 24 hours; lane E, the combination ofsafingol (50 μM) and MMC (5.0 μg/ml) for 24 hours. The results indicatethat untreated gastric cancer cells (lane B) have increased cdk2activity and that both MMC (lane C) and safingol (lane D) have decreasedcdk2. However, the combination of MMC and safingol increased cdk2activity back to basal levels.

These results may explain why safingol, as a single agent, does notinduce apoptosis, but for apoptosis to proceed in the presence ofsafingol there is an obligate requirement for concomitant exposure ofthe tumor cells to a chemotherapeutic agent (e.g.MMC). Applicantspropose that apoptosis takes place when at least three conditions aremet: DNA damage by chemotherapy, an increase in cdk2 activity, andinhibition of PKC activity. Thus, in control cells there is an increasein cdk2 activity but no DNA damage or PKC inhibition; in MMC treatedcells there is DNA damage, a decrease in cdk2 activity, and no PKCinhibition (data not shown); and in safingol treated cells there is PKCinhibition but a decrease in cdk2 activity and no known DNA damage.However, with the combination therapy of safingol and MMC, all threeconditions are met: increased cdk2 activity, DNA damage, and inhibitionof PKC. Thus, apoptosis proceeds. The basis for the decrease in cdk2activity by safingol (as well as by MMC) remains unknown and stands incontrast to the effects of UCN-01 and bryostatin as single agents.However, flavoperidol alone has been reported to inhibit the cdks (39),including cdk2 (Edward Sausville, NCI, Bethesda, Md.), at concentrationsin which applicants have observed flavopiridol to inhibit PKC activity.All these results strongly suggest that cdk2 activity may be a majormarker for the apoptosis associated with PKC inhibition. The increase incdk2 activity with safingol and MMC, indicates a potential biomarker ofapoptosis that will be tested in human tissues on applicants' clinicaltrials (see METHODS and PLANS below).

Safingol enhances the anti-tumor effect of cisplatin and doxorubicin intumor-bearing animals

Although safingol showed no direct anti-tumor activity in vivo, whentested as a single agent in a variety of murine tumor models and humantumor xenografts, its synergism with cisplatin and doxorubicin in vitrosuggested it might potentiate the antitumor efficacy of thesechemotherapeutic agents in vivo. For these experiments safingol wasadministered alone or in combination with cisplatin or DOX to femaleC3H/HeJ mice bearing 16C mammary carcinoma implanted intramuscularly.Antitumor response to the agents alone or in combination was assessed bytumor growth delay. Safingol by itself at doses of 5 to 20 mg/kg had noeffect on inhibiting tumor cell growth. However, when administeredintraperitoneally (i.p.) at a dose of 20 mg/kg and combined withintravenous (i.v.) cisplatin (10 mg/kg), safingol produced a modestenhancement in growth delay when compared to cisplatin alone. Theadministration of cisplatin under these conditions resulted in a thedevelopment of a 16/C mammary carcinoma that took 10 days to reach 5times its starting size, whereas the combination of the same dose ofcisplatin and a single dose resulted in a tumor mass that took 26 daysto reach 5 times its starting size. Comparable effects against 16/Ctumors were observed with safingol and DOX (10 mg/kg, i.v.) (74).

Safingol does not induce increased toxicity of chemotherapy in animals

From the preclinical data it appears that doses of safingol ranging from10 to 20 mg/kg are necessary in order to achieve any level ofchemopotentiation. Because safingol is being developed as achemopotentiation agent, studies were conducted in rats and dogs toevaluate the ability of safingol to potentiate the toxicity ofestablished cancer chemotherapeutic agents (40). Single-dose studieswith rats in which safingol was administered prior to cisplatin,cyclophosphamide, or doxorubicin showed that safingol did not potentiatethe myelotoxicity of these agents or affect reversal of the toxicity. Noovert potentiation of cisplatin-mediated nephrotoxicity was observed.Cisplatin had no apparent effect on the pharmacokinetic profile ofsafingol. A possible effect of safingol on cisplatin pharmacokineticswas demonstrated by a statistically significant higher cisplatin Cmaxvalue following safingol treatment, compared with the cisplatin Cmaxvalue following vehicle administration. Other than the Cmax time point,the concentration-time profile of cisplatin was unaffected by safingol.A single-dose combination study in dogs indicated that safingol did notpotentiate doxorubicin toxicity and vice versa, and that plasmaconcentrations of safingol or doxorubicin were also unaffected byadministration of the other (Investigators brochure).

Intravenous administration of safingol in emulsion caused clinicalpathology and histopathology findings consistent with venous irritationand transient intravascular hemolysis in mice, rats, and dogs (40). Itseemed to occur as a function of safingol concentration and depended onthe caliber of the vein used for delivery. Although venous irritationand hemolysis were significant and, in some instances, dose limitingtoxicities in the animal studies, it was demonstrated that these effectsmay be mitigated by the use of low safingol concentrations andlarge-sized, high flow veins.

Results from the phase I clinical trial combining safingol withdoxorubicin:

(i) Study design: This Phase I trial was designed as an open-label,non-randomized, dose escalation study, in which groups of three to sixpatients received on day 1 of each cycle received sequentially increaseddosages of intravenous safingol emulsion and then (provided there was noacute toxicity) 14 days later received the same dose of safingol onehour prior to a fixed intravenous dose of doxorubicin. Individualpatients could receive up to six cycles of treatment with the same doseof safingol and doxorubicin (DOX) every 21 days, until signs of tumorprogression or unacceptable toxicity occurs, or until the patient'scumulative life-time dose of doxorubicin exceeds 400 mg/m² with anadditional cycle of treatment. Prior DOX therapy was allowed up to adose of 280 mg/m² as long as the left ventricular ejection fractionwas >50%. The starting dose of safingol was 15 mg/m². This represented1/10 the LD₁₀ in mice, 4% the highest non-toxic dose (HNTD) in dogs, and12% the HNTD in rats.

(ii) Patient characteristics: To date applicants have entered 17patients onto the study, all of whom are evaluable. The patientcharacteristic are as follows: 1) median age (range): 59 (29-77), 2)median KPS (range): 80 (70-90) 3) Male:Female : 10:7, 4) Primary sitesof disease: pancreas:6, gastric:2, colon:3, unknown primary:2,sarcoma:3, nasopharyngeal:1;

(iii) No dose limiting hematologic Toxicity: As shown in the table belowwith a fixed doxorubicin dose of 45 mg/m² and escalating doses ofsafingol, there has been no-dose limiting hematologic toxicity. In factthe white blood count nadirs have increased with increasing safingoldose. Dose escalation above 120 mg/m² is planned.

    ______________________________________                                        Hematologic Toxicity with Safingol and DOX at 45 mg/m.sup.2                                                Mean                                                               Mean White Absolute Mean                                                      Blood      Neutrophil                                                                             Platelet                                Safingol                                                                              # of      Count      Count    Count                                   (mg/m.sup.2)                                                                          patients  (range)    (range)  (range)                                 ______________________________________                                        15      3         3.2 (1.4-  1.2 (0.8-                                                                              150 (121-                                                 5.0)       1.8)     189)                                    30      4         4.6 (1.1-  2.3 (0.4-                                                                              187 (37-                                                  9.0)       4.0)     349)                                    60      3         4.0 (2.2-  2.3 (0.7-                                                                              154 (70-                                                  6.1)       4.7)     233)                                    120     3         5.4 (4.9-  5.2 (2.3-                                                                              283 (225-                                                 7.5)       5.1)     279)                                    ______________________________________                                    

b) No dose-limiting hemolysis: Thus far only one patient has experienceda grade I hemolysis as evidenced by a >50% decrease in serumhaptoglobin. This was associated with no changes in reticulocyte count,no evidence of hemolysis on the peripheral smear, no "pink"discoloration to the plasma, and no evidence or urine hemosiderin. Thiswas believed to be related to relatively poor venous access which onlybecame apparent on the first day of therapy. The patient was retreatedaccording to applicants' protocol criteria for grade I hemolysis whichincluded infusion of safingol through a central vein and at one half theconcentration (twice the total volume but the same total dose). Underthese conditions the patient has experienced no further evidence ofhemolysis.

(iii) Safingol pharmacokinetics appear linear with increasing dose: Thepharmacokinetics of patients treated with safingol and DOX at 45 mg/m²is shown below:

    ______________________________________                                        Mean Plasma Safingol Pharmacokinetics with 45 mg/m.sup.2 DOX                  Dose                AUC. (ng ×                                                                        AUC. (ng ×                                Level,    Cmax,     hr/mL)    hr/mL)                                          mg/m.sup.2                                                                              ng/ml     with DOX  without DOX                                     ______________________________________                                        15 (n = 3)                                                                              199       168       161                                             30 (n = 4)                                                                              336       501       428                                             60 (n = 3)                                                                              422       531       501                                             120 (n = 3)                                                                             988       1227      1226                                            ______________________________________                                    

It appears that with the safingol dose escalation the increases inplasma safingol levels is generally linear with increasing safingoldose. In addition, DOX did not change the pharmacokinetics of safingol.

Based on the mice studies applicants have predicted that in order toachieve meaningful chemopotentiation with either DOX or cisplatinapplicants need to deliver the equivalent mouse dose of 5 mg/kg to 20mg/kg saf ingol. From the preclinical pharmacology in the mice thistranslates into a Cmax and AUC of 1,797 ng/ml and 668 ng×hr/mlrespectively for 5 mg/kg safingol and a Cmax and AUC of 7,021 ng/ml and2,702 ng×hr/ml respectively for 20 mg/kg safingol. From the pharmacologydata obtained thus far from the patients treated with escalating dosesof safingol and 45 mg/m² of DOX, it appears that applicants areapproaching these target levels. In view of this and the fact thatapplicants are still significantly below the predicted pharmacologicaltoxicity levels, applicants continue to accrue patients to the study.

METHODS and PLAN

To develop an integrated clinical program with PKC inhibitors incombination with chemotherapy that uses laboratory correlates (PKCactivity, cdk2 activity, and terminal deoxynucleotidyl transferase (TdT)activity in tumor tissues and leukocytes) as putative surrogateend-points of activity. The immediate goal will be to complete twocurrent phase I clinical trials with the PKC specific inhibitorL-threo-dihydrosphingosine (safingol) in combination with (i)doxorubicin (DOX) and (ii) cisplatin with the plan to take this to aphase II study in gastric cancer; and to (iii) initiate additionalclinical trials with combinations of chemotherapy and other PKCinhibitors including UCN-01, flavopiridol, and bryostatin.

Applicants now have two open MSKCC IRB-approved phase I studies withsafingol. The first combination study with doxorubicin (see PRELIMINARYRESULTS) will close once applicants reach the MTD. Based on thepre-clinical data in gastric cancer cells a phase II study of safingoland doxorubicin will then be planned. In the interim the second studywith safingol and cisplatin is soon to open. This study has beenapproved for funding as an administrative supplement by the NCI to theMSKCC P30 CA 08748-30. The study design and plan are as follows:

(i) To complete the phase I trial of safingol and doxorubicin.

(ii) To initiate a phase I study of safingol and cisplatin:

Study design: This Phase I trial is designed as an open-label,non-randomized, dose escalation study, in which groups of three to sixpatients will receive sequentially increased dosages of intravenoussafingol emulsion, one hour prior to a fixed, 75 mg/m² intravenous doseof cisplatin, until dose limiting toxicity is demonstrated in at leastthree of six patients. Based on the current phase I study of safingoland doxorubicin, the starting dose for safingol in this new combinationtrial with cisplatin will be 60 mg/m². The dose of safingol will befixed within each co-hort. Individual patients will receive additionalcycles of treatment with the same dose of safingol and cisplatin every21 days, until signs of tumor progression or unacceptable toxicityoccurs.

iii) To initiate additional clinical trials with other PKC inhibitorsincluding UCN-01, flavopiridol, and bryostatin.

a) A combination study of UCN-01 and Carboplatin: Based on applicants'pre-clinical data with UCN-01 as a PKC inhibitor that enhances theinduction of chemotherapy induced apoptosis. The proposal for this studyis that UCN-01 will be given on day 1 as a 24 hour infusion andcarboplatin be given as 300 mg/m² bolus six hours after initiatingUCN-01. UCN-01 will be escalated in subsequent co-horts but the dose ofcarboplatin will remain fixed. This treatment will be repeated every 28days.

b) Future combination trials of PKC inhibitors (i.e bryostatin) andchemotherapy: For the future studies utilizing PKC inhibitors incombination with chemotherapy, applicants will use a general approach,as outlined below, to their development and conduct. These points,including biostatistical considerations, are also applicable to all thePKC inhibitory studies now in progress or proposed.

Patient eligibility and verification of eligibility: Patients will beselected from applicants' Department of Medicine populations. Forgeneral Phase I studies, all types of cancer will be included includinggastric cancer, other gastrointestinal cancer, non-small cell lungcancer, refractory breast cancer, renal cell cancer, melanoma,gynecologic neoplasms, and sarcomas. This type of design allows us to doPhase I testing of highly specific new agents and utilizes the greatdepth of the MSKCC patient population. Memorial Sloan-Kettering CancerCenter has filed form HHS 441 (Re: Civil Rights), form HHS 641 (Re:Handicapped individuals) and form 639A (re: sex discrimination). Inselecting patients for study in the proposed contract work, due noticeis taken of the NIH/ADAMHA Policy concerning women and minorities inclinical research populations. The study population will be fullyrepresentative of the whole range of patients seen at Memorial Hospital.Unless a disease is gender specific, no eligibility limitations will beemployed regarding age, gender, childbearing potential, race or ethnicorigin.

Applicants' past record indicates a commitment to entering patientsrelatively early in the course of basically incurable advanced neoplasms(e.g., colorectal, renal cells, non small cell lung cancer). As nosystemic therapy offers substantial benefit for such patients,applicants feel that Phase I therapy--especially studies to evaluatecombinations seeking to enhance the activity of active drugs (e.g., MMCand Carboplatin) is the best therapy for many patients with thesediseases. Measurable or evaluable tumor masses or parameters, whiledesirable, will not be required for eligibility.

Protocol Design: All Phase I PKC studies will be performed within thecontext of a specific written protocol. Each individual protocol will beinitially designed after receipt of complete preclinical efficacy,toxicologic and pharmacologic information and discussions with NCIstaff. A biostatistician (Ying Huang, Ph.D) is a member of the PKC PhaseI Group and provides statistical methodology when it is necessary todeviate from the standard dose escalations. Particular attention will begiven to blending applicants' Center's expertise with the needs of theNCI Phase I program. Applicants envision continued efforts towarddeveloping combination therapy programs designed to modulatecytotoxicity and overcome resistance, using PKC as a novel drug target.

Initial Drug Dosage: In combined Phase I trials initial dose scheduleswill utilize known pharmacologic data on each drug, prior clinicalexperience using each drug individually (if available) and thebiochemical rational for combining the drugs. Whenever possible, basiclaboratory data will be used to determine sequence, schedule, modes ofadministration, target plasma levels, etc. However, applicants remaincognizant that modulating agents may not only alter the duration andintensity of the toxic effects of the anticancer agent they are pairedwith but the modulating agents themselves may have toxicities which mustbe taken into account in the phase I design.

Dose Escalation: The scheme of dose escalation is a key feature of PhaseI trials. Historically, a traditionally empiric dose escalation has beenemployed with 3 patients in each dose cohort. For such empiric designsat MSKCC, the following scheme will be utilized: N, 2N, 4N, 8N; thenescalation by increments of 30%. If any toxicity>grade 2 (except nauseaand vomiting>grade 3) is seen early in the escalation, 30% increments ofdrug escalation will be begun from that level causing>grade 2 toxicity.Studies which combine modulators with chemotherapy must be designeddifferently than traditional Phase I studies. The design of these trialsdepends heavily on the support of laboratory investigators to establishtarget of modulation and to monitor the effects. For modulating agents,the goal is to administer the minimum dose of modulator whichconsistently results in the desired modulatory effects. In general, forthe PKC inhibitor being escalated, applicants will choose a startingdose which is known to be without toxicity in previous human experienceand will escalate from this dose in a conservative manner whilemaintaining the dose of the other agents constant.

Pharmacokinetics: Whenever possible, pharmacokinetics will be performedon both the modulator and the chemotherapeutic agent to establish theappropriate schedules for optimal combination. The biological endpointmust be ascertained for each patient to establish the appropriatemodulator dose. Then, the chemotherapy dose must be escalated toidentify the alterations in toxicity. In parallel, pharmacokinetics ofboth agents must be monitored to insure that drug-drug interactions donot materially alter the pharmacokinetic behavior of the two agents.Careful attention will be paid to the biostatistical design and analysisof the trial in order to discriminate the effects of each of theindividual agents from those related to drug-drug interaction.Applicants believe that it is important to incorporate pharmacokineticstudies into applicants' Phase I trials. Applicants will performpharmacokinetic analysis on 2-3 patients at the first level of a trialand at each subsequent level (assuming that the drug is detectable atthe early levels). If the mouse pharmacokinetics are known (i.e., mouseLD₁₀ AUC) and if the pharmacokinetics of the drug can be consideredlinear, then a prediction of the "target" AUC at the human MTD can bemade based on the mouse AUC, and the escalations necessary to reach theprojected MTD can be assessed. Continued pharmacokinetic analysis duringthe course of the trial will allow us to monitor progress toward the MTDand allow us to carefully monitor the levels near the projected MTD. Ifapplicants find that the MTD estimated by the AUC analysis will occurbefore level 4, applicants can amend applicants' protocol to approachthe projected MTD more slowly, escalating by increments of 30%.

Biostatistical considerations: At least 3 study drug-naive patients ateach level shall be necessary to fully evaluate that dose level. When adose level with any patient experiencing Grade 3 or greater drug-relatedtoxicity has been identified, at least 6 patients at that dose levelwill be required to more fully evaluate the nature of that toxicity. TheMTD will be defined as that dose which produces reversible≧grade 3toxicity, exclusive of nausea and vomiting, in 2/6 patients treated.

The probability of dose escalation based on the true incidence of doselimiting toxicity at a specific dose level is shown in the table below.

True Toxicity risk 0.10 0.20 0.30 0.40 0.50 0.60

Probability of Escalation 0.91 0.71 0.49 0.31 0.17 0.08

Thus, the risk of escalation in high toxicity risk dose levels is lessthan 50% for true toxicity rates below 0.30.

One of the principal objectives of Phase I studies will be determinationof the maximum tolerated dose schedule. As outlined above, the treatmentdose will be increased after three patients have been treated at a givendose with no toxicity and further testing will take place at doses withtoxicity seen in the first three patients. Because of the iterativenature of the process to determine MTD, the confidence limit around thetrue toxicity rate will be based on additional patients tested at theMTD. The following table gives the 90% confidence limits for the truetoxicity rate as a function of the number of additional patients treatedat the MTD and the number that experience toxicity.

    ______________________________________                                        # Additional                                                                            # with                                                              Patients  Toxicity,                                                           at the dose                                                                             0       1      2     3   4     5   6                                ______________________________________                                        6         .30     .58    .73   .85 .93   .99                                            0       .009   .06   .15 .27   .42                                  8         .31     .47    .60   .71 .81   .89 .95                                        0       .006   .05   .11 .19   .29 .40                              10        .26     .39    .51   .61 .70   .78 .85                                        0       .005   .04   .09 .15   .22 .30                              12        .22     .34    .44   .53 .61   .68 .75                                        0       .004   .03   .07 .12   .18 .24                              ______________________________________                                    

Thus, if 10 patients are treated at the MTD, and if for instance 2patients have toxicity, there is a 90% chance that the true toxicityrate is between 4 and 51%. It is anticipated that most studies willrequire the accumulation of data on approximately 20-30 adult patients.

(iii) To perform correlative laboratory studies: Studies fromapplicants' laboratory with safingol, as well as studies utilizing otherPKC inhibitors, indicate that the critical event for enhancement ofchemotherapy induced apoptosis by PKC inhibition takes place at the G₁/S interphase and that this is associated with increased cdk2 activity.Therefore, applicants' plan is to measure cdk2 activity, PKC activity,and apoptosis by TdT on tumor tissues and leukocytes obtained frompatients on the clinical trials. For these studies applicants willobtain serial tumor biopsies and peripheral lymphocytes from allpatients on the studies at three time points: a baseline before therapy,following treatment with the PKC inhibitor alone, and followingtreatment with the PKC inhibitor and chemotherapy. Lymphocytes will beobtained from peripheral blood by density gradient centrifugation overFicoll, as described (41). Tumor cells will be carefully dissected fromthe biopsies so as to avoid contamination with normal tissue. Whenavailable tumor cells will be obtained from ascitic fluid with passagethrough a 30 μm nylon mesh, as described (42).

Cdk2 assays: For cdk2 assays tissues and cells will be lysed in LysisBuffer containing 0.1% Tween-20, PMSF (0.2 mM), aprotinin (10 μg/ml),and leupeptin (10 μg/ml). Immunoprecipitation will be performed with 200μg of protein lysate for each kinase reaction (38). Following incubationfor 1 hour on ice with the cdk2 antibody the mixture will beequilibrated with protein A-beads (20-25 μl per immunoprecipitate). Thiswas then rocked for 45-60 minutes in the cold room. The beads will bewashed with Lysis Buffer and Hepes-KOH Kinase Buffer containing 50 mMbeta-glycerophosphate. The kinase reaction mixture will contain 0.2 μgof histone type-I, 300 μM ATP, the kinase buffer, and ³² P!ATP. For eachsample a control sample will be run with the same reaction mixture, butIgG will be used in place of histone Type-I. This allows for detectionon non-specific kinase activity which is then subtracted out from thecdk2 activity to determine the absolute radioactive incorporationattributable to cdk2 activity. Each reaction will be allowed to proceedat 30° for 30 minutes and then the samples will be transferred to a 10%SDS PAGE gel for analysis.

PKC assays: For these studies tissues and cells will be homogenized withgentle ultrasonication. PKC assays will be performed as described (37),but myelin basic protein will be used as a substrate. Briefly, PKC willbe extracted from the gastric cancer cells as described (21,61) using a20 mM Tris-HCL (pH 7.5) buffer containing 2 mM EDTA, 2 mM EGTA, 5 mMβ-mercaptoethanol, 0.5 mM phenylmethylsulphonyl fluoride, 10 ug/mlleupeptin, and 0.25M sucrose. Cells or tissues will be homogenized withbrief sonication. The cell homogenates will then centrifuged at100,000×g for 1 hour at 4° C. The supernatant represents the cytosolicfraction and the pellet the membrane fractions. The pellet isresolubilized in the original Tris-HCl buffer. Both fractions are thenassayed for PKC activity for 15 minutes at 25° C. by determining thetransfer of γ-phosphate group of adenosine-5'-triphosphate to myelinbasic protein (Gibco Laboratory) in a 50 mM Tris-HCl (pH 7.5) buffercontaining 8 mM % L-α-phosphatidylserine, 24 ug/ml PMA, and 12 mMcalcium acetate. The reaction mixture is then transferred to WhatmanP-81 paper for liquid scintillation counting.

TdT assays: Fresh tissue will be fixed in 10% neutral buffered formalinin a coplin jar for 10 minutes at room temperature and then fixed withethanol acetatic acid. For paraffin-embedded tissues, samples will firstbe deparaffinized with ethanol and then protein digested with ProteinaseK. For the TdT assays, the ApopTag Kit (Oncor, Gaithersburg, Md.) isused (5,43). This method employs a fluoresceinated antidigoxigeninantibody directed against nucleotides of digoxigenin-11-dUTP (d-dUTP)which are catalytically added to the 3-OH ends of fragmented DNA by TdT.Briefly, fixed sections are washed and fixed with 1% paraformaldehyde.The fixed cells are incubated in a reaction mixture containing TdT andd-dUTP for 30 minutes at 37° C. Stop/wash buffer is added, and the cellsare resuspended in 100 μl of fluorescinated anti-digoxigenin antibodyfor 30 minutes at room temperature. The slides are then washed with 0.1%Triton X-100 before they examined with a Olympus BH-2 fluorescencemicroscope equipped with a BH2-DM2U2UV Dich. Mirror Cube filter(Olympus, Lake Success, N.Y.) and scored for fluorescence. For positivecontrols a cytospsin preparation on a silanized slide of humanperipheral lymphocytes stimulated with 1 μM dexamethasone will be used.For negative controls sham staining will be performed substitutingdistilled water for TdT.

In order to exclude an effect from chemotherapy alone, this assays willbe repeated in the final co-hort of patients one level below the MTD.Three patients will first be treated with chemothrapy (i,e cisplatin)alone for cycle 1, followed 28 days later by the combination of safingoland cisplatin for cycle 2. Three other patients will first be treatedwith the combination of safingol plus cisplatin for cycle 1, followed 28days later by the cisplatin alone for cycle The cellular responses willbe determined at each of these dosing visits. After completion of theproposed phase I study applicants will examine the results of theseassays to see whether an association exists between a change in thesebiological assays and clinical response and/or toxicity in the patientstreated. The Wilcoxon rank-sum test will be employed to compareresponders to non-responders (or toxicity vs. no toxicity) in order totest the validity of any possible associations observed in this patientpopulation.

To perform laboratory studies with human gastric, breast, and ovarycancer cells to: i) study a variety of new PKC inhibitors currentlyunder pre-clinical development as inducers of apoptosis in combinationwith chemotherapy; ii) define the optimal conditions in which to combinesafingol, UCN-01, flavoperidol, or bryostatin with chemotherapy in orderto achieve the maximum induction of apoptosis for the purpose ofclinical trial development; and iii) further examine in vitro cdk2 andto identify other related cell cycle dependent proteins associated withinduction of apoptosis and the inhibition of PKC;

(i) To evaluate a variety of new PKC inhibitors in combination withchemotherapy to determine their effect on induction of apoptosis:Selective PKC inhibitors other than safingol and UCN-01 may induce evengreater degrees of apoptosis when combined with chemotherapy againstbreast (MDA-MB-468), gastric (SK-GT and MKN-74) and ovarian (OVCAR)cancer cells. The other PKC inhibitors to be tested for the induction ofapoptosis against the cell lines including a series of Roche inhibitors(to be supplied by Roche Pharmaceuticals, Nutley, N.J.), and bryostatin1 (36), an agent that activates PKC with short exposure but inhibits PKCfollowing prolonged drug exposure by inducing PKC degradation. Apoptosiswill be quantitated by two methods: (1) QFM staining: This methodinvolves staining with bisbenzimide trihydrochloride (Hoescht-33258) ofcondensed chromatin which characterizes the cells undergoing apoptosis(3,5) and (ii) terminal deoxynucleotidyl transferase (TdT) assay whichlabels the 3'-OH ends of DNA fragments in apoptotic cells (5,43).

For QFM determinations, the cells are fixed in 3% paraformaldehyde andincubated at room temperature for 10 minutes. The fixative is removedand the cells are washed with 1× PBS, resuspended in 20 μl 1× PBScontaining only 8 μg/ml of bisbenzimide trihydrochloride (Hoechst#33258), and incubated at room temperature for 15 minutes. Aliquots ofthe cells (10 μl) are placed on glass slides coated with3-amino-propyl-triethoxysilane, and duplicate samples of 500 cells eachwere counted and scored for the incidence of apoptotic chromatincondensation using an Olympus BH-2 fluorescence microscope equipped witha BH2-DM2U2UV Dich. Mirror Cube filter (Olympus, Lake Success, N.Y.).

For the TdT assays, the ApopTag Kit (Oncor, Gaithersburg, Md.) will beused. This method will essentially be identical to the one describedabove for tissue and lymphocyte samples from patients except that afterwashing with 0.1% Triton X-100 the cells will be counterstained withpropidium iodide (PI) solution. Green (d-dUTP labeled DNA strand breaks)and red (PI staining for total DNA content) fluorescence of individualcells are measured on a FACScan flow cytometer (Becton Dickinson, SanJose, Calif.). The resulting bivariate plots enables the detection ofapoptotic events within the cell cycle. The R₁ cursor is set using thecontrol specimen to define normal levels of green fluorescence (i.e.,basal levels of apoptosis). Cells with fluorescence above the R₁ cursorare considered apoptotic. The data from 10,000 cells are collected andanalyzed using CellFit and LYSYS software (Becton Dickinson).

(ii) To define the optimal conditions in which to combine safingol,UCN-01, flavoperidol, and bryostatin with chemotherapy for futureclinical development: Safingol, UCN-01, and flavoperidol are now inphase I clinical trials. Combining these agents with chemotherapeuticagents appears to be the best way to maximize the beneficial effects ofthese drugs as anti-cancer agents against breast cancer cells. However,the optimal way in which to achieve this end remains to be defined.Applicants plan to pursue this issue with a series of studies:

The optimal chemotherapeutic agent(s): The plan is to performexperiments with safingol and UCN-01 in combination with otherconventional chemotherapeutic agents such as cis-platin, Taxol,5-fluorouracil and doxorubicin in order to determine the extent to whichthese agents induce apoptosis with these other agents in the breast(MDA-MB-468), gastric (SK-GT and MKN-74) and ovarian (OVCAR) cells. Themethods to quantitate apoptosis will be by QFM and TdT assays, asdescribed above.

Combinations of PKC inhibitors: Since many of these inhibitors act atdifferent sites of the enzyme (regulatory vs. catalytic), it may bepossible to combine multiple PKC inhibitors together with chemotherapyin order to further enhance the induction of apoptosis. For example, asapplicants have shown, the combination of UCN-01 and safingol with MMCat the conditions described above induce a greater degree of apoptosisthan treatment either PKC inhibitor alone with MMC. Further studiescombining flavoperidol with safingol or with UCN-01 are planned andapoptosis will be quantitated by the QFM and the TdT technique.

The optimal sequence and timing of the PKC inhibitors with thechemotherapeutic agent: The exact sequence and timing of the PKCinhibitor relative to chemotherapy exposure in breast cancer cellremains unknown. For these experiments applicants propose exposing thebreast and gastric cancer cells to safingol (50 μM) for different timeintervals (30 minutes, 1 hour, 3 hours, 6 hours, 18 hours, and 24hours), washing the cells free of drug, and then exposing to MMC 5μg/ml) for 24 hours. The relative induction of apoptosis will then bequantitated as above. Conversely, cells will first be exposed tosafingol for 24 hours, but the MMC exposure will now be limited todifferent time intervals before being assayed for apoptosis.

(iii) To examine in vitro cdk2 and to identify other related cell cycledependent proteins associated with the induction of apoptosis and theinhibition of PKC: The basis for the increase in cdk2 activity bysafingol and MMC in combination remains unknown. It may be related to adecrease in the expression of a cdk2 inhibitor (i.e. p21) or it may berelated to a modification of a phosphorylated site on cdk2 which resultsin its activation. A further understanding of this process may lead tonew surrogate markers of activity. In order to examine this furtherapplicants plan to examine: 1) protein expression of p21 and p27 withimmunoblotting using an enhanced chemiluminescence system using specificantibodies (Santa Cruz Biotechnology, Inc. Santa Cruz, Calif.); 2)differences in protein phosphorylation of cdk2 with ³² P!orthophosphatecell labelling, as previously described, using a cdk2 specific antibodysuitable for immunoprecipitating (36); and 3) correlative studies ofcdk2 activity as measured by the histone H1 kinase assays (38). Controlcells will be treated in the same way as described above except thatstandard media without drug will be used for all incubations.

To determine whether a specific PKC isoform may be a critical target fordrug development in induction of apoptosis with chemotherapy by: (i)testing whether antisense for PKCα in combination with MMC has superioranti-tumor activity against human gastric cancer cells when compared toeither agent alone or to a PKC missense with MMC; (ii) testing other PKCantisense against other PKC specific isoforms in combination withchemotherapy; (iii) studying in gastric cancer cells whether any of thePKC isoforms are being preferentially inhibited during the induction ofapoptosis.

(i) Testing whether antisense for PKCα in combination with MMC hassuperior anti-tumor activity against human gastric cancer cells whencompared to either agent alone or to a PKC missense with MMC:applicants' results indicate that PKC represents a novel target forenhancing the induction of apoptosis by chemotherapy. However, PKC isubiquitous in both normal and malignant tissue. Even though safingol'sspecificity represents a considerable advance in the development of PKCinhibitors especially as they pertain to eventual clinical development,even further specificity would still provide theoretical advantages indrug development.

PKC comprises a multigene family, consisting of at least 13 distinctgenes which have 4 large constant regions separated by 5 variableregions (18,44). A current concept is that the variable regions of these13 PKC isoforms encode isozymes-specific properties (45). These 13 PKCisozymes can be grouped into three categories based on Ca²⁺ requirementsfor activation and phorbol ester binding activity. PKC is also the majorcellular receptor for tumor promoting phorbol esters which, as analoguesof DAG, directly activate PKC (19). These isoforms are designated α, β1,β2, γ, δ, ε, η, θ, μ, ζ, λ, and τ. The biological significance of PKCheterogeneity is unknown, but it is speculated that, since they showdifferential tissue expression, specific PKC isoforms will phosphorylatedifferent substrates and mediate distinct and different biologicalresponses in the cells in which they are expressed. The role of thesedifferent isoforms is essentially unknown. However, identifying theisoform(s) involved in apoptosis would provide a more specific targetfor drug therapy.

Recent data have implicated PKCα in apoptosis. Studies have shown thatif PKCα is inactivated then apoptosis can proceed. Most attempts todevelop isoforms specific PKC inhibitors have been unsuccessful.However, some inroads have been made with PKCα by testing of antisenseoligonucleotides. A 20 mer has been developed by Isis Pharmaceuticals(Carlsbad, Calif. 92008) which specificly inhibits the expression ofhuman PKCα without affecting the expression of other PKC isoforms. Ithas been shown to inhibit the growth of subcutaneously implanted humantumor xenografts (46). This inhibition was dose-dependent whenadministered intravenously (i.v.) or intraperitoneally (i.p.) between0.06 and 6 mg/ml.

Applicants' results indicate that the best way to maximize thetherapeutic benefits of PKC inhibitors is to combine them withchemotherapy. Applicants have previously reported that all the SK-GTcell lines express PKCα mRNA and protein (15,47). Therefore, these celllines appear to be appropriate for the evaluation of a PKCα anti-sense.The goal of this study is to test the combination of the PKCα anti-sensein combination with MMC for the first time in an in vivo system. The PKCinhibitor for these studies will be a 20 mer PKCα antisense. The doseand schedule (10 mg/kg, daily, i.p.) of the anti-sense in the vivo studywill be based on preclinical toxicology, which has been generated byISIS pharmaceutical. The dose appears to be a safe and well-tolerateddose when administered both i.p. or i.v. to tumor bearing nude mice. Theanti-sense easily dissolves in saline. Doses of 100 mg/kg have beengiven to mice on a daily schedule with no major toxicity and no deaths.At the highest non-toxic dose (100 mg/kg) mild lymphocytic infiltrationhas been noted in the liver, spleen and kidney. In order to determinethe specificity of this response, a missense 20 mer will also beadministered to two of the control groups. This will ensure that theobserved response is secondary to the effect of the PKCα anti-sense andnot due to a non-specific effect of the anti-sense therapy. The missenseselected has also been safely administered at 10 mg/kg i.p to mice on adaily schedule without significant toxicity. The dose of MMC selectedfor these studies will be 14.5 mg/kg in saline. This dose has beensafely administered to nude mice and has also been safely administeredto nude mice when combined with the PKC inhibitor UCN-01 (31).

The animal protocol (MSKCC IACUC #95-07-027) design will be as follows:All animals with be injected subcutaneously into the hind quarter on day1 with 5×10⁶ SK-GT-2 cells. Applicants' preclinical; data indicates thatwithin 10-12 days these animals develop a palpable 0.25 gr. tumor mass.Once the animals have a palpable tumor, they will be randomly assignedto one of 4 treatment arms (17 animals in each arm):

(1) PKCα anti-sense: by itself at a dose of 10 mg/kg/day i.p. daily forfour weeks, (2) PKCα anti-sense+MMC: PKC anti-sense will be given at adose of 10 mg/kg i.p daily for two weeks. On the first day of week #3MMC will be given as a single i.v. dose (14.5 mg/kg). PKC anti-sensewill be repeated i.p daily for an additional two weeks (i.e. weeks #3and 4). (3) MMC alone: Daily i.p injections of a saline control for twoweeks. On the first day of week #3 a single dose of Mitomycin-C well beadministered (14.5 mg/kg) i.v.. This will be followed by two additionalweeks of i.p. saline control. (4) PKCα missense: by itself at a dose of10 mg/kg/day i.p. daily for four weeks.

Assuming a "non-treatment" response rate of 5% (i.e. antisense alone orMitomycin-C alone) relative to the control animals (missense alone),then in order to detect a 50% decrease in size of the palpable tumormass for the treated group (i.e antisense+Mitomycin-C) using a one sidedalternative normal approximation z-test with a significance of 0.05 anda power of 80%, the total number of animals for each group needs to be17. The total number of animals will be 64.

(ii) Testing other PKC antisense against other PKC specific isoforms incombination with chemotherapy: If anti-sense for PKCα does not show anenhancement of MMC-induced apoptosis, then anti-sense against the otherPKC isoforms will be tested according to the same study design. Thematerials for these PKC specific anti-sense studies will be supplied byIsis Pharmaceuticals (Carlsbad, Calif.), as become available.

(iii) Studying in gastric cancer cells whether any of the PKC isoformsare being preferentially inhibited during the induction of apoptosis:Even though safingol is not PKC isoform specific, in regard to theinduction of apoptosis safingol may still be inhibiting one isoformpreferentially over another. One way to study this is to examine therelative distribution of the PKC isoforms between the membrane and thecytosolic fractions of the cells. PKC isoforms become activated whenthey are translocated from the cytosol to the membrane of the cell (48).Conversely, inactivation of these isoforms is associated with depletionof the isoform(s) from the membrane component of the cells. In order totest the influence of isoform distribution on chemotherapy inducedapoptosis, applicants propose treating the cell lines with the fourconditions that applicants have used in applicants' prior studies: (i)no drug (control) for 24 hours, (ii) Safingol alone (50 μM) for 24hours, (iii) Mitomycin-C alone (5 μg/ml) for 24 hours, (iv) thecombination of safingol (50 μM) and Mitomycin-C (5.0 μg/ml) for 24hours. Since the distribution of the PKC isoforms between the cytosolicand membrane fraction of the cells appears to be the critical event fortheir relative activation, applicants will obtain cytosolic and membranefractions, as described (13), to determine whether there are differencesin isoform expression and localization with these different drugtreatments that applicants have shown induce apoptosis. Briefly, PKCwill be extracted from the gastric cancer cells, using a 20 mM Tris-HCL(pH 7.5) buffer containing 2 mM EDTA, 2 mM EGTA, 5 mM β-mercaptoethanol,0.5 mM phenylmethylsulphonyl fluoride, 10 ug/ml leupeptin, and 0.25Msucrose (Buffer A). Cells will be homogenized and th homogenates willthen be centrifuged at 100,000×g for 1 hour at 4° C. The supernatantrepresents the cytosolic fraction and the pellet which is resolubilizedin buffer A containing 0.1% Triton-X represents the membrane fraction.These fractions are then passed through a DEAE-52 column and the PKCprotein is eluted off the column with Buffer A containing 0.15M NaCl.Briefly, cells are lysed following drug treatment with lysis bufferscontaining protease and phosphatase inhibitors. Proteins are resolved bySDS-PAGE electrophoresis. Immunoblots are prepared with Immobilon Pmembranes by electrophoretic transfer and blocked in TN-Tween buffer (50mM Tris, pH 7.5, 200 mM NaCl, 0.1% Tween-20) containing 5% (w/v) nonfatdry milk. After washing the blots are incubated with horseradishperoxidase-conjugated secondary antibodies (dilution 1/2000.Immunologically reactive protein will be visualized with the enhancedchemiluminescence system (Amersham, Arlington Heights, Ill.). using PKCisoform specific antibodies (49) (Santa Cruz Biotechnology, Inc. SantaCruz, Calif.).

Reference of the Second Series Experiment

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Third Series of Experiments

Protein Kinase C as a Novel Target for Inducing Apoptosis in BreastCancer

This invention develops new therapeutic approaches in the treatment ofbreast cancer by utilizing protein kinase C (PKC) as a target forinducing apoptosis in breast cancer cells. The specific aims are:

1. To determine the optimal chemotherapeutic agents to combine with thePKC specific inhibitors safingol and UCN-01 in the induction ofapoptosis;

2. To evaluate other novel PKC inhibitors in combination withchemotherapy to determine their effects on induction of apoptosis;

3. To test the optimal combination and sequence of PKC inhibitors withchemotherapy;

4. To determine whether a specific PKC isoform may be a critical targetfor drug development in inducing apoptosis in breast cancer cells;

5. To assess the relationship between PKC and cell cycle regulation inthe induction of apoptosis.

BACKGROUND and SIGNIFICANCE

In the United States adenocarcinoma of the breast is the most commonmalignant neoplasm in women. It is estimated that over 182,000 new casesof breast cancer will be diagnosed in 1995 and this will be associatedwith approximately 46,000 deaths, making breast cancer the secondleading cause of cancer related-death in women in the United States (1).The incidence of breast cancer has been rising steadily in this countryat a rate of one to two percent a year. Even though multi-agentchemotherapy in the adjuvant setting has had a significant impact onimproving overall survival, the five year survival in node-positivebreast cancer following adjuvant chemotherapy still remainsapproximately 65% (2). Furthermore, for metastatic breast cancer,despite innumerable clinical trials using various combinations ofchemotherapy, the survival of patients has not significantly improved(3). Because of the high incidence of breast cancer worldwide, evensmall improvements in the efficacy of treatment may represent improvedsurvival for tens of thousands of patients.

The breast tumors of many of these patients are resistant to the mostactive chemotherapeutic agents. The basis of drug resistance in breastcancer has not been well defined. The expression of the multi-drugresistant gene (mdr1) in breast cancer is low (4), suggesting alternatemechanisms of drug resistance. Recent studies have indicated a linkbetween the induction of apoptosis (programmed cell death) and p53expression (5). Cells which express wild-type p53 are capable ofundergoing apoptosis after exposure to common chemotherapeutic agents orionizing radiation; whereas cells with mutated or deleted p53 areresistant, avoiding apoptosis and continuing to replicate. Similarly, inbreast cancer mutation in p53 has been associated with increasedchemotherapy resistance (6). The incidence of p53 mutation in breastcancer has been reported to be 22% in primary lesions of patients withsporadic carcinomas (7) and may be as high as 50% in lymph nodescontaining metastatic disease (8). For familial syndromes the incidenceranges from 34% for patients with familial breast cancer to 52% inpatients with the familial breast and ovary cancer syndrome (7). In viewof the prevalence of p53 mutations in breast cancer overcoming this formof resistance may greatly enhance the efficacy of cancer chemotherapy.

A large body of evidence indicates a fundamental role for protein kinaseC (PKC), a multigene family of serine/threonine protein kinases, inprocesses relevant to neoplastic transformation, carcinogenesis, andtumor cell metastases (9-12). The activity of PKC has been reported tobe increased in human breast cancer cell lines as (13), well as humanbreast cancer tissue as compared to normal breast tissue from the samepatient (14). Consequently, PKC may represent a novel target foranti-cancer therapy in breast cancer. In order to test this hypothesisthe PKC specific inhibitor safingol (e.g. the L-threo enantiomer ofdihydrosphingosine) has been tested both by itself and in combinationwith conventional chemotherapeutic agents. While safingol as a singleagent had negligible impact on tumor growth of mice-bearing mousemammary 16/C and MT#7 carcinomas, the combination of safingol withdoxorubicin or cisplatin substantially potentiated the anti-tumoreffects of these drugs (15). Based on these observations, safingol, usedin combination with doxorubicin, has become the first PKC specificinhibitor to enter clinical trials (16).

The mechanism by which safingol potentiates the activity ofchemotherapeutic agents is unclear, although inhibition ofP-glycoprotein phosphorylation and reversal of the multidrug resistant(mdr) phenotype in breast cancer cell lines has been suggested (17-18).While this hypothesis can explain the synergism achieved withcombinations of safingol and doxorubicin, it does not explain thesynergism reported for combinations of safingol with drugs that are notbelieved to produce resistance by the mdr mechanism (e.g., cisplatin)(15), nor does it explain safingol-induced effects that occur in tumorcell lines that do not express the P-glycoprotein (18). Therefore,pathways other than P-glycoprotein inhibition are likely to be involvedin the safingol-mediated enhancement of chemotherapy.

It has been suggested that the anti-tumor activity of manychemotherapeutic agents (e.g., cisplatin and etoposide) is a consequenceof their induction of apoptosis (5,19). In this context it has beenproposed that activation of PKC acts as an antagonist to apoptosis,whereas inhibition of PKC promotes apoptosis (20-22). Thus,safingol-mediated potentiation of chemotherapy might be attributed toits PKC inhibitory effect, subsequently leading to increased apoptosisafter drug-induced damage. In order to test this hypothesis applicantsinitially sought to determine the extent to which safingol by itself, orin combination with a specific chemotherapeutic drug (e.g. mitomycin-C,MMC), would promote apoptosis in gastric cancer cells. Furthermore,applicants investigated whether the p53 status of these cells influencesthe development of apoptosis after treatment with safingol and MMC (23).

For these initial studies applicants used the gastric cancer cell linesSK-GT-5 cells, which have a mutated p53 gene and are resistant to MMC,and MKN-74, which are wild-type for p53 and sensitive to MMC (23).Neither cell line expresses P-glycoprotein. Applicants' results showthat safingol alone did not induce apoptosis in either the MMC-sensitiveMKN-74 cells, or the MMC-resistant SK-GT-5 cells, as quantified by bothbisbenzimide trihydrochloride staining and the TdT assay (24). Inaddition, the typical oligonucleosomal base pair fragments (DNA"ladders") were not induced by safingol under the conditions tested.When exposed to MMC alone, apoptosis was induced in both cell linesalthough to different degrees. However, addition of safingolsignificantly potentiated this apoptotic response. For MKN-74 cells MMCinduced apoptosis in 40% of the cells, whereas the combination of MMCand safingol induced apoptosis in 83% of the exposed cells. With theSK-GT-5 cells, MMC alone induced apoptosis in 18% of the cells, whereasthe combination of safingol and MMC induced apoptosis in 39% of thecells (24). These studies showed that safingol potentiated the cytotoxiceffect of MMC in two human gastric cancer cell lines which differed intheir baseline sensitivity to MMC and in their p53 status. Applicantshave observed a similar potentiation of apoptosis by safingol incombination with other chemotherapeutic agents, including doxorubicin,against the gastric cancer cells.

In order to test whether the effect of safingol in inducing apoptosiswas due to its anti-PKC effect, applicants performed comparable studieswith safingol in the presence of the phorbol ester, 3-phorbol12-myristate 13-acetate (PMA), which activates PKC by binding to itsamino-terminal regulatory domain (25). Safingol, as a sphingosine,inhibits PKC activity by interfering with the function of PKC'sregulatory domain (26). Therefore, applicants hypothesized that if thepotentiation of MMC-induced apoptosis by safingol is mediated byinhibition of PKC, then PMA should abrogate this effect. In applicants'studies with the gastric cancer cells, PMA effectively abrogated thesafingol effect of potentiating MMC-induced apoptosis in this cells,supporting the hypothesis that this process is a PKC-dependent event(22).

Applicants' studies demonstrate that safingol sensitizes gastric cancercells, with either wild-type or mutated p53, to the induction ofapoptosis by MMC. Applicants therefore sought to determine whether thiseffect by safingol on MMC induced apoptosis could be detected in breastcancer cells.

PRELIMINARY STUDIES

For these studies applicants used MDA-MB-468 breast cancer cells whichhave a mutation in p53 (7) and have PKC enzyme activity, as determinedby enzyme extraction on a DEAE-52 column and assayed for PKC activity byincorporation of γ³² P!ATP into myelin basic protein. For determinationof apoptosis MDA-MB-468 cells were treated according to one of severalconditions: (i) no drug (control) for 24 hours, (ii) Safingol alone (50μM) for 24 hours. (This concentration represents the highest non-toxicdose of safingol for the MDA-MB-468 cells, as determined by cellproliferation studies, and slightly exceeds the safingol concentration(30 μM) which inhibits PKC enzyme activity by 50% in vitro.), (iii)Mitomycin-C alone (2.5 μg/ml) for 24 hours, (iv) Mitomycin-C alone (5μg/ml) for 24 hours, (v) the combination of safingol (50 μM) andMitomycin-C (2.5 μg/ml) for 24 hours, (vi) the combination of safingol(50 μM) and Mitomycin-C (5 μg/ml) for 24 hours. Apoptosis was measuredby quantitative fluorescent microscopy (QFM) of nuclear changes inducedby apoptosis, as determined by bisbenzimide trihydrochloride(Hoescht-33258) staining of condensed nuclear chromatin (24). For theQFM duplicate samples of 500 cells each were counted and scored for theincidence of apoptotic chromatin condensation using an Olympus BH-2fluorescence microscope. The results are shown in FIG. 9. MMC alone atthe concentrations of 2.5 μg/ml and 5.0 μg/ml induced apoptosis in 4%±2and 6%±1 of the MDA-MB-468 cells, respectively. However, the combinationof safingol (SPC) and MMC significantly increased the percentage ofcells undergoing apoptosis from 11%±1 with safingol alone to 23%±2 withsafingol and 2.5 μg/ml MMC and to 33%±10 with safingol and 5.0 μg/ml MMC(p<0.001).

Applicants also tested the PKC inhibitor UCN-01 (27) which is currentlyin phase I clinical trial at the NCI. In contrast to safingol, whichinhibits PKC by binding to its regulatory domain, UCN-01 inhibits PKC atits catalytic domain. Therefore, assessment of the effect of UCN-01 oninducing apoptosis in combination with MMC on MDA-MB-468 was alsoperformed. The QFM method to quantitate apoptosis was again used. Theconditions tested were identical to those described above for safingolexcept UCN-01 (1 μM) was substituted for safingol in all the conditions.The results are shown in FIG. 10. The combination of UCN-01 and MMCtogether increased the induction of apoptosis of the MDA-MB-468 cellsfrom 20%±4 with UCN-01 alone to 41%±3 with UCN-01 and 2.5 μg/ml MMC andto 58%±1 with UCN-01 and 5.0 μg/ml MMC.

These results indicate that even though the PKC inhibitors safingol andUCN-01 can by themselves induce a slight degree of apoptosis in thebreast cancer cells the induction of apoptosis is greatly enhanced whenthe PKC inhibitors are given in combination with MMC. The demonstrationthat these agents potentiate chemotherapy induced apoptosis may haveimportant clinical implications in view of the recent introduction ofthese two agents into clinical trials. Of particular importance is thefinding that these agents are effective even with tumor cells that havea mutation in p53. Hence the use of PKC inhibitors may provide a newapproach to overcoming drug resistance in tumor cells that are resistantto chemotherapy because they lack p53 function. The purpose of thesestudies is then to determine how to optimize this effect in breastcancer cells by examining the best combinations of PKC inhibitors withchemotherapy as well as to examine the cellular basis of thisphenomenon.

METHODS and PLAN

To determine the optimal chemotherapeutic agent(s) to combine with thePKC specific inhibitor safingol and UCN-01 in the induction ofapoptosis. Both Safingol and UCN-01 are now in phase I clinical trials.Combining these agents with chemotherapeutic agents appears to be thebest way to maximize the beneficial effects of these drugs asanti-cancer agents against breast cancer cells. However, the optimalchemotherapeutic agent to combine with safingol and UCN-01 againstbreast cancer cells remains to be defined. The plan is to performstudies with safingol and UCN-01 in combination with other conventionalchemotherapeutic agents such as cis-platin, Taxol, 5-fluorouracil anddoxorubicin in order to determine the extent to which safingol andUCN-01 induce apoptosis with these other agents. For these studiesapplicants will use MDA-MB-468 cells. Applicants will also test otherbreast cancer cell lines, including MCF-7 and MCF-7 sublines resistantto doxorubicin and cisplatin, in order to determine whether thisphenomenon of enhancing the induction of apoptosis with PKC inhibitorsis more generalized. Apoptosis will be quantitated by two methods: (1)QFM staining: This method involves staining with bisbenzimidetrihydrochloride (Hoescht-33258) of condensed chromatin whichcharacterizes the cells undergoing apoptosis (24), and (ii) terminaldeoxynucleotidyl transferase (TdT) assay which labels the 3'-OH ends ofDNA fragments in apoptotic cells (24,28).

For QFM determinations, the cells are fixed in 3% paraformaldehyde andincubated at room temperature for 10 minutes. The fixative is removedand the cells are washed with 1× PBS, resuspended in 20 μl 1× PBScontaining only 8 μg/ml of bisbenzimide trihydrochloride (Hoechst#33258), and incubated at room temperature for 15 minutes. Aliquots ofthe cells (10 μl) are placed on glass slides coated with3-amino-propyl-triethoxysilane, and duplicate samples of 500 cells eachwere counted and scored for the incidence of apoptotic chromatincondensation using an Olympus BH-2 fluorescence microscope equipped witha BH2-DM2U2UV Dich. Mirror Cube filter (Olympus, Lake Success, N.Y.).

For the TdT assays, the ApopTag Kit (Oncor, Gaithersburg, Md.) is used.This method employs a fluoresceinated antidigoxigenin antibody directedagainst nucleotides of digoxigenin-11-dUTP (d-dUTP) which arecatalytically added to the 3-OH ends of fragmented DNA by TdT. Briefly,1-2×10⁶ cells were washed and fixed with 1% paraformaldehyde. The fixedcells are incubated in a reaction mixture containing TdT and d-dUTP for30 minutes at 37° C. Stop/wash buffer is added, and the cells areresuspended in 100 μl of fluorescinated anti-digoxigenin antibody for 30minutes at room temperature. The cells are then washed with 0.1% TritonX-100 and counterstained with propidium iodide (PI) solution. Green(d-dUTP labeled DNA strand breaks) and red (PI staining for total DNAcontent) fluorescence of individual cells are measured on a FACScan flowcytometer (Becton Dickinson, San Jose, Calif.). The resulting bivariateplots enables the detection of apoptotic events within the cell cycle.The R₁ cursor is set using the control specimen to define normal levelsof green fluorescence (i.e., basal levels of apoptosis). Cells withfluorescence above the R₁ cursor are considered apoptotic. The data from10,000 cells are collected and analyzed using CellFit and LYSYS software(Becton Dickinson).

To evaluate other novel PKC inhibitors in combination with chemotherapyto determine their effect on induction of apoptosis. Selective PKCinhibitors other than safingol and UCN-01 may induce even greaterdegrees of apoptosis when combined with chemotherapy against breastcancer cells. The other PKC inhibitors to be tested for the induction ofapoptosis against the breast cancer cells will include RO 32-0432 (29),a specific inhibitor of PKC's catalytic domain (supplied by RochePharmaceuticals, Nutley, N.J.), and bryostatin 1 (30), an agent thatactivates PKC with short exposure but inhibits PKC following prolongeddrug exposure by inducing PKC degradation. The methods to quantitateapoptosis will be similar to those described above.

To test the optimal combination and sequence of PKC inhibitors withchemotherapy. Since many of these inhibitors act at different sites ofthe enzyme (regulatory vs. catalytic), it may be possible to combinemultiple PKC inhibitors together with chemotherapy in order to furtherenhance the induction of apoptosis. For example, applicants plan todetermine whether the combination of UCN-01 and safingol with MMC at theconditions described above induce a greater degree of apoptosis thantreatment either PKC inhibitor alone and MMC. In addition, the exactsequence and timing of the PKC inhibitor relative to chemotherapyexposure in breast cancer cell remains unknown. For these experimentsapplicants propose exposing the MDA-MB-468 cells to safingol (50 μM) fordifferent time intervals (30 minutes, 1 hour, 3 hours, 6 hours, 18hours, and 24 hours), washing the cells free of drug, and then exposingto MMC 5 μg/ml) for 24 hours. The relative induction of apoptosis willthen be quantitated as above. Conversely, cells will first be exposed tosafingol for 24 hours, but the MMC exposure will now be limited todifferent time intervals before being assayed for apoptosis.

To determine whether a specific PKC isoform may be a critical target fordrug development in inducing apoptosis in breast cancer cells; PKCcomprises a multigene family, consisting of at least 13 distinct genesdesignated α, β, γ, δ, ε, ζ and L (7). The β gene actually yields twodistinct transcripts designated β₁ and β₂. These isoforms showdifferential tissue expression, exhibit differences in enenzymaticproperties, and may carry out different specialized functions. Theirrole in apoptosis remains unknown. Since PKC is expressed to some degreein even normal breast tissue (14), it would be ideal to identify PKCisoforms that are unique to human breast tumors. Identifying such anisoform and directly relating the expression of this isoform to theinduction of apoptosis in breast cancer cells would provide a strongrationale for drug development of isoform specific inhibitors in thisdisease. Already some progress has been made in this area with thedevelopment of antisense oligonucleotides. A 20 mer has been developedthat specifically inhibits the expression of human PKCα withoutaffecting the expression of other PKC isoforms (31). It has also beenshown, as a single agent, to inhibit growth of subcutaneously implantedhuman tumor xenografts. Recent data has, in fact, implicated PKCα inapoptosis of bovine aortic endothelial cells. These studies indicatethat only if PKCα is inhibited (e.g defined in these studies asinability to translocate from the cytosol to the active membranefraction) can apoptosis proceed (22).

In order to pursue this further in breast cancer cells, applicants firstpropose identifying those isoforms that are expressed in the MDA-MB-468cells and determining whether the PKC inhibitors (i.e safingol andUCN-01) affect their protein expression in the presence or absence ofMMC. Since the distribution of the PKC isoforms between the cytosolicand membrane fraction of the cells appears to be the critical event fortheir relative activation, applicants will obtain cytosolic and membranefractions, as described (13), to determine whether there are differencesin isoform expression and localization with the different drugtreatments that applicants have shown induce apoptosis.

Briefly, PKC will be extracted from the breast cancer cells, using a 20mM Tris-HCl (pH 7.5) buffer containing 2 mM EDTA, 2 mM EGTA, 5 mMβ-mercaptoethanol, 0.5 mM phenylmethylsulphonyl fluoride, 10 ug/mlleupeptin, and 0.25M sucrose (Buffer A). Briefly cells will behomogenized with a 80 strokes (the minimum number required to break thebreast cancer cells) of a hand-held Dounce homogenizer. The cellhomogenates will then be centrifuged at 100,000×g for 1 hour at 40° C.The supernatant represents the cytosolic fraction and the pellet whichis resolubilized in buffer A containing 0.1% Triton-X represents themembrane fraction. These fractions are then passed through a DEAE-52column and the PKC protein is eluted off the column with Buffer Acontaining 0.15M NaCl. Briefly, cells are lysed following drug treatmentwith lysis buffers containing protease and phosphatase inhibitors.Proteins are resolved by SDS-PAGE electrophoresis. Immunoblots areprepared with Immobilon P membranes by electrophoretic transfer andblocked in TN-Tween buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 0.1%Tween-20) containing 5% (w/v) nonfat dry milk. After washing the blotsare incubated with horseradish peroxidase-conjugated secondaryantibodies (dilution 1/2000. Immunologically reactive protein will bevisualized with the enhanced chemiluminescence system (Amersham,Arlington Heights, Ill.). using PKC isoform specific antibodies (SantaCruz Biotechnology, Inc. Santa Cruz, Calif.).

To assess the relationship between PKC and the cell cycle: The resultswith gastric cancer cells indicate that p53 is not critical to theinduction of apoptosis with PKC inhibition since this phenomenon isobserved in the presence of tumor cells with wild type and mutant p53.The preliminary studies with the breast cancer cell line MDA-MB-468,which is known to contain a mutation in p53, would support thishypothesis and would indicate that the effect of PKC inhibitors onenhancing MMC-induced apoptosis is independent of the p53 status of thecells. The existence of a p53-independent pathway for growth arrest hasbeen reported (32). Further studies to define the effects of safingol,especially as it pertains to PKC and steps independent of p53 in thecell cycle of breast cancer cells are proposed. Preliminary data fromapplicants' lab would indicate that the critical event for induction ofapoptosis by PKC inhibition takes place at the G1/S interphase. Proteinsimplicated in regulating this phase of the cell cycle are the cyclinkinases, including cyclin dependent kinase 2, and the cyclin kinaseinhibitors, including p21 and p27. In order to magnify the effect oftreatment on these events in the cell cycle, the MDA-MB-468 cells willbe synchronized with nocadozole (0.2 μg/ml) for 18 hours, washedthoroughly for 4 hours, and then exposed to 50 μM safingol for 24 hoursin the presence or absence of MMC (5 μg/ml). Control cells will betreated in the same way as described above except that standard mediawithout drug will be used for all incubations.

The plan is to examine the effect of safingol and chemotherapy (i.e MMC)on the expression and activation of these proteins by: 1) proteinexpression with immunoblotting using an enhanced chemiluminescencesystem; 2) differences in protein phosphorylation with ³²P!orthophosphate cell labelling, as previously described (33); 3) andcyclin dependent kinase activity as measured by the histone H1 kinaseassays (34).

The protein kinase C(PKC) inhibitors UCN-01 and flavopiridol (Flavo)significantly enhance the cytotoxic effect of chemotherapy by promotingapoptosis in gastric and breast cancer cells.

We have reported that the PKC inhibitor safingol significantly enhancesthe cytotoxic effects of Mitomycin-C (MMC) by promoting MMC-inducedapoptosis in gastric cancer cells (JNCI 87: 1394, 1995). Both UCN-01 andFlavo inhibit PKC. These drugs are cytotoxic to tumor cells and havebeen reported to induce apoptosis. Our studies with safingol indicatethat the effectiveness of UCN-01 and Flavo on inducing apoptosis wouldbe greatly enhanced by combining them with chemotherapy. In order totest this hypothesis we elected to treat gastric cancer cells, MKN-74and SK-GT-2, as well as breast cancer cells, MDA-MB-468, with UCN-01(7.5 to 10 μM) or Flavo (300 nM) in the presence or absence of MMC (5μg/ml) or Taxol (50 μM) for 24 hours. Induction of apoptosis wasestimated by counting the frequency of condensed nuclear chromatin withHoechst-33258 stain in duplicate samples of 400 cells. The results aresummarized as follows:

    ______________________________________                                        Cell Line No drug  MMC      Flavo  Flavo + MMC                                ______________________________________                                        MKN-74    1% ± 1                                                                              7% ± 1                                                                              17% ± 2                                                                           73% ± 1, p<.001                         ______________________________________                                                                           UCN-01 +                                   Cell Line No drug  Taxol    UCN-01 Taxol                                      ______________________________________                                        MKN-74    1% ± 1                                                                              2% ± 1                                                                              10% ± 2                                                                           28% ± 9, p<.05                          ______________________________________                                        Cell Line No drug  MMC      UCN-01 UCN-01 + MMC                               ______________________________________                                        SK-GT-2   1% ± 1                                                                              11% ± 2                                                                             18% ± 4                                                                           53% ± 1, p<.001                         MDA-MB-468                                                                              1% ± 1                                                                              6% ± 1                                                                              20% ± 1                                                                           58% ± 1, p<.001                         ______________________________________                                    

These results indicate that even though both UCN-01 and Flavo can induceapoptosis as single agents their greatest effect is observed when theyare combined with chemotherapy. These studies suggest that combinationsof these agents with classical chemotherapeutic drugs may improve theefficacy of chemotherapy in both gastric and breast cancer.

References of the Third Series of Experiments

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2. Hortobagyi G. N., and Buzdar A. U. Current status of adjuvantsystemic therapy for primary breast cancer: progress and controversy. CA45: 199-226.

3. Schwartz G. K., and Bhardwaj S. Chemotherapy of breast cancer In:Gunter Deppe (ed.): Chemotherapy of gynecologic cancer, second edition:303-362, 1990.

4. Goldstein L. J., Galski H., Fojo A., Willingham M., Lai S. -L.,Gazdar A., et al. Expression of a multidrug resistance in human cancers.J. Natl. Cancer Inst. 81:116-24, 1989.

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7. Thor A. D., Moore D. H., Edgerton S. M., Kawasaki E. S., Reihsaus E.,Lynch H. T. et al. Accumulation of p53 tumor suppressor gene protein: anindependent marker of prognosis in breast cancers. J. Natl. CancerInstitute 84: 845-55, 1992.

8. Davidoff A. M., Kerns B. J., Pence J. C., Marks J. R., and IglehartJ. D. p53 alterations in all stages of breast cancer. J. Surg. Oncol.48: 260-267, 1991.

9. Nishizuka Y. The role of PKC in cell surface signal transduction andtumor promotion Nature, 308: 693-698, 1984.

10. Housey G. M., Johnson M. D., Hsiao W. L. W., O'Brian C. A., MurphyJ. P., Kirschmeirer P., and Weinstein I. B. Overproduction of proteinkinase C causes disordered growth control in rat fibroblasts. Cell 52:343-354, 1988.

11. Schwartz, G. K., Redwood S. M., Ohnuma T., Holland J. F., Droller M.J., and Liu B. C. S. Inhibition of invasion of invasive human bladdercarcinoma cells by protein kinase C inhibitor staurosporine. J. Natl.Cancer Inst. 82: 1753-1756, 1990.

12. Schwartz G. K., Jiang J., Kelsen D. P. and Albino A. P. Proteinkinase C: a novel target for inhibiting gastric cancer cell invasion. J.Natl. Cancer Inst., 85: 402-407, 1993.

13. Schwartz G. K., Arkin H., Holland J. F. and Ohnuma T. Decreasedprotein kinase C activity and multidrug resistance in MOLT-3 humanlymphoblastic leukemia cells resistant to trimetrexate. Cancer Res., 51:55-61, 1991.

14. O'Brian C., Vogel V. G., Singletary S. E., and Ward N. E. Elevatedprotein kinase C expression in human breast tumor biopsies relative tonormal breast tissue. Cancer Res. 49: 3215-3217, 1989.

15. Adams L. M., Dykes D., Harrison S. D., Saleh J., and Saah L.Combined effect of the chemopotentiator SPC-100270, a protein kinase Cinhibitor, and doxorubicin or cisplatin (Cis) on murine isografts andhuman tumor xenografts. Proc. Amer. Assoc. Cancer Res. 34:410, 1993.

16. Schwartz G. K., Ward D., Saltz L. Casper E., et al. A phase I studyof the protein kinase C specific inhibitor safingol alone and incombination with doxorubicin. Proc. Amer. Soc. Clin. Onc. 14:1557,1995.

17. Sachs C. W., Safa A., and Fine R. L. Inhibition of protein kinase Cby Safingol is associated with chemosensitization of multidrug resistantMCF-7 cells. Proc. Amer. Assoc. Cancer Res. 35:447, 1994.

18. Adams L. M., Cofield D. J., Seldin J. C., Kitchen P. A., et al.Effect of the protein kinase C (PKC) inhibitor SPC-100270 on drugaccumulation and cytotoxicity in drug resistant and sensitive tumor cellin vitro. Proc. AACR 34:410, 1993.

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20. Jarvis W. D., Turner A. J., Povirk L. F., Traylor R. S., and GrantS. Induction of apoptotic DNA fragmentation and cell death in HL-60human promyelocytic leukemia cells has been reported for pharmacologicalinhibitors of protein kinase C. Cancer Res. 54: 1707-1714, 1994.

21. McConkey D. J., Hartzell P., Jondal M., and Arrenius S. Inhibitionof DNA fragmentation in thymocytes and isolated thymocyte nuclei byagents that stimulate protein kinase. J. Biol. Chem. 264: 13399-13402,1989.

22. Haimovitz-Friedman A., Balaban N. A., McLoughlin M., Ehleiter D.,Michaeli J., Vlodavsky I., and Fuks Z. PKC mediates basic fibroblastgrowth factor of endothelial cells against radiation-induced apoptosis.Cancer Res. 54: 2591-2597,1994.

23. Nabeya Y., Loganzo F., Maslak P., Lai L., de Oliveira A., SchwartzG. K. et al. The mutational status of p53 protein in gastric andesophageal adenocarcinoma cell lines predicts sensitivity tochemotherapeutic agents. Int. J. Cancer 64: 1-10, 1995.

24. Schwartz G. K., Haimovitz-Friedman A., Dhupar S. K., Ehleiter D.,Maslak P., Loganzo F., Kelsen D. P., Fuks Z., Albino A. P. Potentiationof apoptosis by treatment with the protein kinase C specific inhibitorsafingol in mitomycin-c treated gastric cancer cells. J. Natl. CancerInstitute 87(18): in press, 1995.

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28. Schmitz G., Walter T., Serbel R., and Kessle C. Nonradioactivelabeling of oligonucleotides in vitro with the hapten digoxigenin bytailing with terminal transferase. Anal. Biochem. 192: 222-231, 1991.

29. Birchall A. M., Bishop J., Bradshaw D., Cline A., Coffey J., ElliottL. H., et al. RO 32-0432, a selective and orally active inhibitor ofprotein kinase C prevents T-cell activation. J. Pharmacol. Exp.Therapeutics, in press, 1995.

30. Kraft A. S., Smith J. B., and Berkow R. L. Bryostatin, an activatorof calcium phospholipid-dependent protein kinase, blocks phorbolester-induced differentiation of human promyelocytic HL-60 cells. Proc.Natl. Acad. Sci. USA 83:1334-1338,1986.

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Fourth Series of Experiments

To evaluate the ability of the PKC inhibitor RO 32-2241 to increase thesensitivity of gastric cancer cells to the cytotoxic effects ofMitomycin-C.

Gastric cancer is one of the leading causes of cancer death throughoutthe world. The reported 5 years survival of surgically resected patientsapproaches 15-20% while the overall survival of patients with gastriccarcinoma is approximately 10%. Currently there are no effectiveregimens for inhibiting the growth gastric cancer. Therefore, theidentification of an agent which inhibits the growth of gastric cancermay have a significant clinical impact on prolonging the survival ofpatients with this disease.

A large body of evidence indicates a fundamental role for theinvolvement of protein kinase C (PKC), a family ofserine/threonineprotein kinases, in processes related to neoplastic transformation,carcinogenesis, and tumor cell invasion. Consequently, PKC may present anovel target for anti-cancer therapy. RO 32-2241, is abisindolymaleimide that inhibits PKC by binding to its catalytic site.Introduction of a cationic side chain and conformational restriction ofthe amine side chain has resulted in an agent that is highly selectivefor PKC and which can penetrate cells. Preclinical animal studies showthat RO 32-2241 is non-toxic at doses that achieve serum levelssufficient to inhibit PKC-driven responses. For example, this agent hasbeen shown to be an inhibitor of T cell activation and phorbol esterinduced paw edema in female AHH/R rats. However, despite extensiveevaluation of this agent as an anti inflammatory drug, there has been noformal testing of this agent in cancer therapy.

It has been shown that PKC activity can act as an antagonist toapoptosis, whereas PKC inhibition can promote apoptosis. Theseobservations imply that one function of PKC stimulated processes is toinhibit the induction of programmed cell death. Thus, anti-tumor effectsof chemotherapeutic agents may be potentiated by PKC inhibitors thatcontribute to the induction of apoptosis after drug-induced damage. Inorder to test this hypothesis we have used the human gastric cancer cellMKN-74 to determine whether RO 32-2241 would enhance the cytotoxiceffect of the chemotherapeutic agent Mitomycin-C (MMC). Cytotoxicity wasmeasured and quantified using the Alamar Blue Assay. Cells were treatedaccording to one of four conditions: (i) no drug (control) for 24 hours,(ii) RO 32-2241 alone at 10-6M, (iii) Mitomycin-C alone at 5 μg/ml for24 hours, (iv)the combination of RO 32-2241 (10-6M) and Mitomycin-C(5μg/ml) for 24 hours. The results are summarized below for percentsurvival:

    ______________________________________                                        Drug Treatment      Percent survival                                          ______________________________________                                        MMC                 87%                                                       RO 32-2241 alone    100%                                                      Combination of MMC + RO 32-2241                                                                   42%                                                       ______________________________________                                    

Thus, treatment of these cells with either RO 32-2241 or MMC alone hadessentially minimal or no effect on inhibiting cellularproliferation;whereas the combination of these two agent at the sameconcentrations was exceptionally toxic to the MKN-74 cells.

In contrast to safingol, the PKC inhibitor currently in clinical atMSKCC, RO 32-2241 is a more protent and selective inhibitor of PKC.Whereas safingol inhibits PKC in micromolar ranges, RO 32-2241 inhibitsPKC activity in nanomolar ranges. Therefore, in view of itsexceptionally potency and specificity there are theoretical advantagesfor the development of RO 32-2241 for clinical trial. The purpose of thepresent study therefore is to determine whether effect that we areobserving with RO 32-2241 in combination with mitomycin-C in vitro canalso be achieved in tumor bearing animals in vivo.

The PKC inhibitor for these studies will be RO 32-2241. The dose andschedule of the PKC inhibitor for this study (30 mg/kg), i.p./day, ×4weeks) is based on preclinical toxicology, which has been generated byROCHE Research center (Welwyn Garden City, UK). The dose appears to be asafe and well-tolerated dose when administered i.p. to MF1 mice. It hasbeen shown to effectively inhibit PKC-mediated responses (e.g paw edema)in animals. This effect has been shown to persist for up to 6 hours whenRo 32-2241 is administered either 1 or 24 hours before challenge with aninflammatory stimulus. The inhibitor will be formulated by sonicating in10% succinylated gelatin to form a solution appropriate for i.p.injection. This will be freshly prepared every 5 days. (The 10%succinylated gelatin is prepared by slowly dissolving in distilledwater, stirring continuously, and heated below 60° C. One prepared it isautoclaved and stored below 5° C.) Doses of 200 mg/kg/day have beengiven to mice on this schedule with no major animal toxicity or deaths.

The decision to combine RO 32-2241 with MMC is based on our pre-clinicaldata which indicated that RO 32-2241 enhanced the cytotoxicity of MMC.The dose of MMC selected for these studies will be 4 mg/kg in saline.The dose has been safely administered to nude mice and has also beensafely administered to nude mice when combined with the PKC inhibitorUCN-01 (Akinaga et al., Enhancement of anti-tumor activity of mitomycinC in vitro and in vivo by UCN-01, a selective inhibitor of proteinkinase C. Cancer Chem Pharm 32: 183-189, 1993).

The protocol design will be as follows: All animals with be injectedsubcutaneously into the hind quarter on day 1 with 5×10⁶ MKN-74 cells.Our preclinical data indicates that within 10-12 days these animalsdevelop a palpable 0.25 gr. tumor mass. Once the animals have a palpabletumor, they will be randomly assigned to one of 4 treatment arms (17animals in each arm) on day #1:

1) Ro 32-2241: by itself at a dose of 30 mg/kg/day i.p. starting day #1for 4 weeks.

2) MMC alone: administered alone (4.0 mg/kg) i.v., x1, day #1.

3) RO 32-2241+MMC: Ro32-2241 will be given at a dose of 30 mg/kg/dayi.p. starting day #1 and repeated daily for 4 weeks. One hour followingthe day #1 i.p. injection of RO 32-2241, MMC will be given as a singlei.v. dose (4.0 mg/kg)

    ______________________________________                                        Group                                                                         (17     RO 32-2241   Mitomycin-C                                              animals/                                                                              (30 mg/kg/day, i.p.,                                                                       (4.0 mg/kg, × 1,                                   group)  × 4 weeks)                                                                           day #1)     Saline controls                              ______________________________________                                        I       +            -           -                                            II      -            +           -                                            III     +            +           -                                            IV      -            -           +                                            (control)                                                                     ______________________________________                                    

Following the final dose of RO 32-2241 each treatment arm will bedivided into 3 groups (5 animals/subgroup) and each subgroup will thenbe used for 0 hr, 1 hr, and 3 (or 6) hour time points for bloodcollection following the final dose of RO 32-2241. Blood samples will becollected into EDTA tubes.

Plasma will be separated by centrifugation (2000 g; 10 min, at 4° C., ifpossible) within 2 hours of collection and stored at -20° C.

Fifth Series of Experiments

Evaluation of the Protein Kinase C α Anti-sense, Both by Itself and inCombination with Mitomycin-C, as a New Anti-Cancer Treatment for GastricCancer

To evaluate the anti-tumor activity of the PKC α anti-sense both byitself and in combination with mitomycin-C against gastric cancer.

A large body of evidence indicates a fundamental role for theinvolvement of protein kinase C (PKC), a family of serine/threonineprotein kinases, in processes related to neoplastic transformation,carcinogenesis, and tumor cell invasion. Consequently, PKC may present anovel target for anti-cancer therapy. It has been shown that PKCactivity can act as an antagonist to apoptosis, whereas PKC inhibitioncan promote apoptosis. These observations imply that one function of PKCstimulated processes is to inhibit the induction of programmed celldeath. Thus, anti-tumor effects of chemotherapeutic agents may bepotentiated by PKC inhibitors that contribute to the induction ofapoptosis after drug-induced damage.

In order to test this hypothesis applicants have used gastric cancercells (MKN-74 and SK-GT-5) to determine the induction of apoptosis withthe PKC inhibitor safingol alone and in combination with thechemotherapeutic agent Mitomycin-C. Safingol is ideal for theseexperiments since it specificly inhibits PKC activity by binding to theenzymes's regulatory domain, a site which is unique to PKC. This is incontrast to other PKC inhibitors such as staurosporine which inhibit PKCenzyme activity by binding to the enzyme's catalytic domain, a site thatis highly homologous to the catalytic domain of other protein kinases.

For these studies apoptosis was measured by quantitative fluorescentmicroscopy (QFM) determination with bisbenzimide trihydrochloride stain(Hoescht-33258) for condensed nuclear chromatin characteristic ofapoptotic cells. Gastric cancer cells (MKN-74) were treated according toone of several conditions: (i) no drug (control) for 24 hours, (ii)safingol alone at 50 μM, the highest non-toxic dose as determined bycell proliferation studies with ³ H!-thymidine for 24 hours; (iii)Mitomycin-C alone (5 μg/ml) for 24 hours; (iv) the combination ofsafingol (50 μM) and Mitomycin-C (5 μg/ml) for 24 hours. The doses ofMMC used was based on cell proliferation studies with ³ H!-thymidinewith gastric cancer cells that indicate<20% inhibition of proliferation.

For QFM determinations 500 cells were counted and scored for theincidence of apoptotic chromatin changes under an Olympus BH-2fluorescence microscope in duplicate samples, ±SD, using a BH2-DM2U2UVDich. Mirror Cube filter. The results were summarized in FIG. 2.

As the results indicate treatment of MKN-74 cells with safingol alonedid not induce apoptosis in these cells. However, the combination ofsafingol with Mitomycin-C induced an increase in apoptosis in adose-dependent fashion when compared to treatment with Mitomycin-Calone. The percentage of cells undergoing apoptosis increased from 40%with Mitomycin-C alone to 80% with the combination therapy. This effectwas observed in cells with wild type (MKN-74) or mutated (SK-GT-5) p53indicating that this effect was independent of the p53 status of thecells (Schwartz GK, et al, Potentiation of apoptosis by treatment withthe protein kinase C specific inhibitor safingol in Mitomycin-C treatedgastric cancer cells, JNCI, In press). Applicants have obtained similarresults with other human gastric cancer cell line that includingSK-GT-1, SK-GT-4, SK-GT-5. Each of these cell lines has a mutation inp53 and exhibits drug resistance to chemotherapy (including MMC). Thus,PKC inhibitors appear to represent a novel way to enhance chemotherapysensitivity even in tumor cells that are resistant to chemotherapy byvirtue of their p53 mutational status.

These results indicate that PKC represents a novel target for enhancingthe induction of apoptosis when combined with chemotherapy. However, PKCis ubiquitous in both normal and malignant tissue. Even though safingolrepresents a considerable advance in the development of PKC inhibitorsespecially as they pertain to eventual clinical development, furtherspecificity would still provide theoretical advantages in drugdevelopment. PKC exists as a multi-gene family with multiple isoformsthat are both tumor and site specific. The role of these differentisoforms is essentially unknown. However, identifying the isoform(s)involved in apoptosis would provide a more specific target for drugtherapy. Recent data have implicated PKCα in apoptosis. Studies haveshown that if PKCα is inactivated then apoptosis can proceed(Haimovitz-Friedman et al, Protein kinase C mediates basic fibroblastgrowth factor protection of endothelial cells against radiation-inducedapoptosis, Cancer Res. 54: 2591-2597, 1994).

Most attempts to develop PKC isoform specific inhibitors have beenunsuccessful. However, some inroads have been made with PKCα by testingof antisense oligonucleotides. A 20 mer has been developed by IsisPharmaceuticals (Carlsbad, Calif. 92008) which specificly inhibits theexpression of human PKCα without affecting the expression of other PKCisoforms. It has been shown to inhibit the growth of subcutaneouslyimplanted human tumor xenografts. This inhibition was dose-dependentwhen administered intravenously (i.v.) or intraperitoneally (i.p.)between 0.06 and 6 mg/ml (Dean, N. et al. Inhibition of growth ofxenografted human tumor cell lines in nude mice by an antisenseoligonucleotide targeting human PKCα. Proc. AACR 36: 2460, 1995.)

Applicants' results would indicate that the best way to maximize thetherapeutic benefits of PKC inhibitors is to combine them withchemotherapy. Applicants have previously reported that all the SK-GTcell lines express PKCα mRNA and protein (Schwartz G. K., et al.Defining the invasive phenotype of proximal gastric cancer cells. Cancer73: 22-27, 1994). Therefore, these cell lines appear to be appropriatefor the evaluation of a PKCα anti-sense. Applicants' published resultson the effect of combining Mitomycin-C with PKC inhibitors is based onin vitro systems. Ultimately, applicants' observation needs to beverified in an in vivo model. Therefore, the goal of this study is totest the combination of the PKCα anti-sense in combination with MMC forthe first time in an in vivo system.

Gastric cancer is one of the leading causes of cancer death throughoutthe world. The reported 5 years survival of surgically resected patientsapproaches 15-20% while the overall survival of patients with gastriccarcinoma is approximately 10%. Currently there are no effectiveregimens for inhibiting the growth of gastric cancer. Therefore, theidentification of an agent which inhibits the growth of gastric cancermay have a significant clinical impact on prolonging the survival ofpatients with this disease.

The PKC inhibitor for these studies will be a 20 mer PKCα antisense. Thedose and schedule of the anti-sense for this study (10 mg/kg, daily,i.p.) will be based on preclinical toxicology, which has been generatedby ISIS pharmaceutical. The dose of appears to be a safe andwell-tolerated dose when administered both i.p. or i.v. to tumor bearingnude mice. The anti-sense easily dissolves in saline. Doses of 100 mg/kghave been given to mice on a daily schedule with no major toxicity andno deaths. At the highest non-toxic dose (100 mg/kg) mild lymphocyticinfiltration has been noted in the liver, spleen and kidney.

In order to determine the specificity of this response, a missense 20mer will also be administered to two of the control groups. This willensure that the observed response is secondary to the effect of the PKCαanti-sense and not due to a non-specific effect of the anti-sensetherapy. The missense selected has also been safely administered at 10mg/kg i.p to mice on a daily schedule without significant toxicity.

The decision to combine PKCα antisense with MMC is based on applicants'pre-clinical data which indicated that PKC inhibitors enhanced theinduction of MMC-induced apoptosis. The dose of MMC selected for thesestudies will be 14.5 mg/kg in saline. This dose has been safelyadministered to nude mice and has also been safely administered to nudemice when combined with the PKC inhibitor UCN-01 (Akinaga et al.,Enhancement of anti-tumor activity of mitomycin C in vitro and in vivoby UCN-01, a selective inhibitor of protein kinase C, Cancer Chem Pharm32: 183-189, 1993).

The protocol design will be as follows: All animals with be injectedsubcutaneously into the hind quarter on day 1 with 5×10⁶ SK-GT-2 cells.Applicants' preclinical; data indicates that within 10-12 days theseanimals develop a palpable 0.25 gr. tumor mass. Once the animals have apalpable tumor, they will be randomly assigned to one of 4 treatmentarms (17 animals in each arm):

1) PKCα anti-sense: by itself at a dose of 10 mg/kg/day i.p. daily forfour weeks,

2) PKCα anti-sense+MMC: PKC anti-sense will be given at a dose of 10mg/kg i.p daily for two weeks. On the first day of week #3 MMC will begiven as a single i.v. dose (14.5 mg/kg). PKC anti-sense will berepeated i.p daily for an additional two weeks (i.e. weeks #3 and 4).

3) MMC alone: Daily i.p injections of a saline control for two weeks. Onthe first day of week #3 a single dose of Mitomycin-C well beadministered (14.5 mg/kg) i.v.. This will be followed by two additionalweeks of i.p. saline control.

4) PKCα missense: by itself at a dose of 10 mg/kg/day i.p. daily forfour weeks.

Because of the nature of anti-sense therapy, in vitro studies are notpredictive of in vivo results. For this reason applicants plan to godirectly to these animal studies.

Athymic nu/nu mice are ideal recipients of human tumor cells becausethey will not usually show host versus graft reactions and thusimmunologically attack the injected tumor cells.

    ______________________________________                                                 PKCα             PKC                                                    antisense              missense                                      Group    (10 mg/kg    Mitomycin-                                                                              (10 mg/kg                                     (17      1.P,         C (14.5   i.p,                                          animals/ daily, ×                                                                             mg/kg,    daily, ×                                group)   28 days)     day #14)  28 days)                                      ______________________________________                                        I        +            -         -                                             II       -            -         -                                             III      +            +         -                                             IV       -            +         +                                             (control)                                                                     ______________________________________                                    

Assuming a "non-treatment" response rate of 5% (i.e. antisense alone orMitomycin-C alone) relative to the control animals (missense alone),then in order to detect a 50% decrease in size of the palpable tumormass for the treated group (i.e antisense+Mitomycin-C) using a one sidedalternative normal approximation z-test with a significance of 0.05 anda power of 80%, the total number of animals for each group needs to be17. The total number of animals will be 64.

Sixth Series of Experiments

Structural modification of protein kinase C specific inhibitors predictsfor enhancement of mitomycin-C induced apoptosis in gastric cancer cells

Applicants have reported that the protein kinase C (PKC) specificinhibitor safingol enhances mitomycin-C (MMC)-induced apoptosis ingastric cancer cells (JNCI 87:1394, 1995). To study this furtherapplicants elected to examine five bis-indolylmaleimides with variouspotencies against PKC. Unlike safingol, which inhibits PKC at itsregulatory domain, these compounds inhibit PKC by competing with ATP atits catalytic domain. MKN-74 gastric cancer cells were treated for 24hours according to one of four conditions: 1) No drug, 2) PKC inhibitor(1 to 4 μM), 3) MMC alone (5 μg/ml, 4) MMC+PKC inhibitor. The inductionof apoptosis was estimated by counting the frequency of condensednuclear chromatin with Hoechst-33258 stain in duplicate samples of 400cells. None of the PKC inhibitors induced a significant degree ofapoptosis when given alone. However, only two compounds (RO-A and RO-B)enhanced MMC-induced apoptosis from 10%±2% with MMC alone to 41%±1% withthe combination of RO-A and MMC (p<0.001) and to 34%±2% with thecombination of RO-B and MMC (p<0.001). Interestingly, other compounds,one with similar potency against PKC (RO-C), did not potentiateMMC-induced apoptosis. Therefore, potent activity against PKC appears tobe necessary, but not sufficient, to cause apoptosis in combination withMMC. Examination of the structure of the two active PKC inhibitorsindicates a common modification of the indole ring which the other PKCinhibitors lack. These results suggest that this specific modificationof bisindolylmaleimides, which confers PKC specificity, has thepotential to enhance chemotherapy-induced apoptosis.

What is claimed is:
 1. A method for screening for protein kinase Cinhibitors which increase induction of apoptosis in tumors cellscomprising the steps of:(a) contacting tumor cells with an amount of aprotein kinase C inhibitor effective to increase induction of apoptosisof the tumor cells; (b) contacting the tumor cells of step (a) with anantitumor therapeutic agent; (c) determining the apoptosis of the tumorcells of step (b); and (d) comparing the apoptosis determined in step(c) with the apoptosis of control tumor cells which are only treatedwith the antitumor therapeutic agent, a greater degree of apoptosisdetermined in the tumor cells in step (c) than in the control tumorcells indicating that the protein kinase C inhibitor increases inductionof apoptosis in those tumor cells.
 2. A method for screening for proteinkinase C inhibitors which increase induction of apoptosis in tumorscells comprising the steps of:(a) contacting tumor cells with an amountof a protein kinase C inhibitor in the presence of an antitumortherapeutic agent effective to increase induction of apoptosis of thetumor cells; (b) determining the apoptosis of the tumor cells of step(a); and (c) comparing the apoptosis determined in step (b) with theapoptosis of control tumor cells which are only treated with theantitumor therapeutic agent, a greater degree of apoptosis determined inthe tumor cells in step (b) than in the control tumor cells indicatingthat the protein kinase C inhibitor increases induction of apoptosis inthose tumor cells.
 3. A method according to claim 1 or 2, wherein theprotein kinase C inhibitor is selected from the group consisting ofprotein kinase C inhibitors and antisense nucleotides capable ofinhibiting the expression of protein kinase C.